Synthetic membrane-receiver complexes

ABSTRACT

Compositions comprising synthetic membrane-receiver complexes, methods of generating synthetic membrane-receiver complexes, and methods of treating or preventing diseases, disorders or conditions therewith.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/738,414, filed Jun. 12, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/581,486, filed Dec. 23, 2014, which is acontinuation of International Application No. PCT/US2014/065304, filedNov. 12, 2014, entitled “Synthetic Membrane-Receiver Complexes”, whichclaims the benefit of U.S. Provisional Application No. 61/962,867, filedNov. 18, 2013; U.S. Provisional Application No. 61/919,432, filed Dec.20, 2013; U.S. Provisional Application No. 61/973,764, filed Apr. 1,2014; U.S. Provisional Application No. 61/991,319, filed May 9, 2014;U.S. Provisional Application No. 62/006,825, filed Jun. 2, 2014; U.S.Provisional Application No. 62/006,829, filed Jun. 2, 2014; U.S.Provisional Application No. 62/006,832, filed Jun. 2, 2014; U.S.Provisional Application No. 62/025,367, filed Jul. 16, 2014; U.S.Provisional Application No. 62/059,100, filed Oct. 2, 2014, all of whichare incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 17, 2015, isnamed 28345US_CRF_sequencelisting.txt and is 67,497 bytes in size.

FIELD OF THE INVENTION

The field of the invention is pharmaceutical compositions for thetreatment of diseases and disorders.

BACKGROUND

The circulatory system permits blood and lymph circulation to transport,e.g., nutrients, oxygen, carbon dioxide, cellular waste products,hormones, cytokines, blood cells, and pathogens to and from cells in thebody. Blood is a fluid comprising, e.g., plasma, red blood cells, whiteblood cells, and platelets that is circulated by the heart through thevertebrate vascular system. The circulatory system becomes a reservoirfor many toxins and pathogenic molecules upon their introduction to orproduction by the body. The circulatory system also serves as areservoir for cellular secretions or detritus from within the body. Theperpetual or aberrant circulation and proliferation of such moleculesand entities can drive disease and/or exacerbate existing conditions.

The efficacy of therapeutic compositions that alleviate or preventdiseases and conditions associated with the circulatory system is oftenlimited by their half-life, which is typically up to a few days. Theshort half-life often necessitates repeated injections andhospitalizations. It is thought that the short half-life may be due toboth renal clearance, e.g., of proteins smaller than 60 kDa, andnon-renal clearance, e.g., via liver excretion or immune-mediatedremoval. The activity of therapies is also often limited by an immunereaction elicited against them (see, e.g., Wang et al., Leukemia 2003,17:1583). Several approaches are practiced in the art.

One approach includes the use of “erythrocyte ghosts” that are derivedfrom a hemolyzed red blood cell. To prepare erythrocyte ghosts, redblood cells undergo hypotonic lysis. The red blood cells are exposed tolow ionic strength buffer causing them to burst. The resulting lysedcell membranes are isolated by centrifugation. The pellet of lysed redblood cell membranes is resuspended and incubated in the presence of thetherapeutic agent, for example, such as an antibiotic orchemotherapeutic agent in a low ionic strength buffer. The therapeuticagent distributes within the cells. Erythrocyte ghosts and derivativesused to encapsulate payloads, such as therapeutic agents, can shieldthose payloads from the immune system, but the erythrocyte ghoststhemselves are subject to rapid clearance by the reticulo-endothelialsystem (see, e.g., Loegering et al. 1987 Infect Immun 55(9):2074).Erythrocyte ghosts also elicit an immune response in mammalian subjects.These vesicles are typically constituted of both lipids and proteins,including potentially high amounts of phosphatidylserine, which isnormally found on the inner leaflet of the plasma membrane. This leadsto potential immunological reactions in the recipient mammaliansubjects. The undesirable effects seriously limit the potential fortherapeutic applications of technologies based on erythrocyte ghosts.

Another approach for drug encapsulation includes the use of exosomes.“Exosomes” include cell-derived vesicles that are present in many andperhaps all biological fluids, including blood, urine, and culturedmedium of cell cultures. The reported diameter of exosomes is between 30and 100 nm, which is larger than low-density lipoprotein (LDL), butsmaller than, for example, red blood cells. Exosomes are either releasedfrom the cell when multivesicular bodies fuse with the plasma membraneor they are released directly from the plasma membrane. Exosome deliverymethods require a better understanding of their biology, as well as thedevelopment of production, characterization, targeting and cargo-loadingnanotechnologies. Attempts have been made to manufacture exosomes usinghuman embryonic stem cell derived mesenchymal stem cells (hESC-MSCs).However, as hESC-MSCs are not infinitely expansible, large scaleproduction of exosomes would require replenishment of hESC-MSC throughderivation from hESCs and incur recurring costs for testing andvalidation of each new batch (Chen et al. 2011 Journal of TranslationalMedicine 9:47). Clinical translation is also hindered by the lack ofsuitable and scalable nanotechnologies for the purification and loadingof exosomes (Lakhal and Wood 2011 BioEssays 33(10):737). Currentultracentrifugation protocols are commercially unreproducible, as theyproduce a heterogeneous mix of exosomes, other cellular vesicles andmacromolecular complexes. Therefore, purification methods based on theuse of specific, desired markers, such as the expression of a targetingmoiety on the surface of the exosome, are required. In addition, siRNAloading into exosomes is relatively inefficient and cost-ineffective,highlighting the need for the development of transfection reagentstailored for nanoparticle applications. Further, exosomes are rapidlycleared from circulation and substantially accumulate in the liverwithin 24 hours of administration (Ohno et al., 2013 Mol Therapy21(1):185), limiting their application for long-term drug delivery tothe circulatory of a subject.

Polyethylene glycol-coated liposomes are presently used as carriers forin vivo drug delivery. A “liposome” includes an artificially-preparedspherical vesicle composed of a lamellar phase lipid bilayer. Theliposome can be used as a vehicle for administration of nutrients andpharmaceutical agents. Liposomes can be prepared by disruptingbiological membranes, e.g., by sonication. Liposomes are often composedof phosphatidylcholine-enriched phospholipids and may also contain mixedlipid chains with surfactant properties such as eggphosphatidylethanolamine A liposome design may employ surface ligandsfor attaching to a target, e.g., unhealthy tissue. Types of liposomesinclude the multilamellar vesicle (MLV), the small unilamellar liposomevesicle (SUV), the large unilamellar vesicle (LUV), and the cochleatevesicle. Liposomes as carriers of anthracycline antibiotics have been asubject of a great number of studies. As a result, liposome formulationsof daunorubicin (DaunoXome™) and doxorubicin (Doxil™) are nowcommercially available. The pharmacokinetics of the liposomal forms ofanthracycline antibiotics differ from that of their free forms in higherpeak concentrations and longer circulations times of the drugs. Thekinetics of DaunoXome and Doxil clearance from plasma is close tomono-exponential. The half-life of DaumoXome in patient plasma is on theorder of a few hours. In Doxil, polyethylene glycol-coated liposomes areused. The immune system poorly recognizes such liposomes; therefore theplasma half-life of Doxil is in the order of tens of hours.

Red blood cells have been considered for use, e.g., to degrade toxicmetabolites or inactivate xenobiotics, as drug delivery systems, ascarriers of antigens for vaccination, and in other biomedicalapplications (Magnani Ed. 2003, Erythrocyte Engineering for DrugDelivery and Targeting). Many of these applications require proceduresfor the transient opening of pores across the red cell membrane. Drugshave commonly been loaded into freshly isolated red blood cells, withoutculturing, using disruptive methods based on hypotonic shock. Hypotonicdialysis can induce a high degree of hemolysis, irreversiblemodifications in the morphology of the cells and phosphotidyl serineexposure, which has been recognized as an important parameter associatedwith premature red blood cells removal and induction oftransfusion-related pathologies (Favretto 2013 J Contr Rel).

Many drugs, particularly protein therapeutics, stimulate immunogenicresponses that include B cell antibody production, T cell activation,and macrophage phagocytosis. The causes of immunogenicity can beextrinsic or intrinsic to the protein. Extrinsic factors are drugformulation, aggregate formation, degradation products, contaminants anddosing. The administration mode, as well as the drug regimen, alsostrongly influences how immunogenicity is assessed. That is,immunogenicity will have different effects for drugs that are given inacute indications compared to drugs to treat chronic diseases. In thelatter case, patients are exposed to the drug over a longer period oftime and as such can mount a complete response. Pegylation is atechnology designed to prolong the half-life, as well as minimizeimmunogenic responses. In contrast to assumptions that polyethyleneglycol (PEG) is non-immunogenic and non-antigenic, certain animalstudies show that uricase, ovalbumin and some other PEGylated agents canelicit antibody formation against PEG (anti-PEG). In humans, anti-PEGmay limit therapeutic efficacy and/or reduce tolerance ofPEG-asparaginase (PEG-ASNase) in patients with acute lymphoblasticleukemia and of pegloticase in patients with chronic gout, but did notimpair hyposensitization of allergic patients with mPEG-modified ragweedextract or honeybee venom or the response to PEG-IFN in patients withhepatitis C. Anti-PEG antibodies can be found in 22-25% of healthy blooddonors. Two decades earlier, the occurrence was 0.2%. This increase maybe due to an improvement of the limit of detection of antibodies and togreater exposure to PEG and PEG-containing compounds in cosmetics,pharmaceuticals and processed food products. These results raiseconcerns regarding the efficacy of PEG-conjugated drugs for a subset ofpatients (Garay, Expert Opin Drug Deliv, 2012 9(11):1319).

Attempts in the art to create passive half-life improvement methodsfocus on increasing the apparent hydrodynamic radius of a drug. Thekidney's glomerular filtration apparatus is the primary site in the bodywhere blood components are filtered, see for reference e.g., Osicka etal. Clin Sci 1997 93:65 and Myers et al. Kidney Int 1982 21:633. Themain determinant of filtration is the hydrodynamic radius of themolecule in the blood; smaller molecules (<80 kDa) are filtered out ofthe blood to a higher extent than larger molecules. Researchers haveused this generalized rule to modify drugs to exhibit a largerhydrodynamic radius and thus longer half-life, mainly via chemicalconjugation to large molecular weight water-soluble polymers, such aspolyethylene glycol (PEG). Numerous PEGylated protein and small moleculetherapeutics are currently offered in the clinic (Pasut and Veronese,2009 Adv Drug Deliv Rev 61(13):1177; Fishburn, 2008 J Pharm Sci97(10):4167). Though effective in many cases in increasing circulationhalf-life, especially as the hydrodynamic radius of the graft or fusionincreases (Gao, Liu, et al., 2009 PNAS 106(36):15231), these methodsoffer challenges in manufacturing and maintenance of biological effectorfunction. Heterogeneities in conjugation reactions can cause complexproduct mixtures with varying biological activities, due mostly to theutilization of site-unspecific chemistries. Extensive biochemicalcharacterization often follows precise purification methods to retain ahomogenous therapeutic product (Huang, Gough, et al, 2009 Anal Chem81(2):567; Bailon, Palleroni, et al., 2001 Bioconj Chem 12(2):195;Dhalluin, Ross, et al., 2005 Bioconj Chem 16(3):504). Furthermore,attachment of large moieties, such as branched PEGs, to reactive zonesof proteins can lead to decreased receptor affinity (Fishburn, 2008 JPharm Sci 97(10):4167).

Albumin may be used to bind a therapeutic protein for increasedcirculation of the drug (Dennis et al, 2002 J Bil Chem 277(38):35035;Walker, Dunlevy, et al., 2010 Prot Engr Des Sel 23(4):271) to increasethe apparent size of the therapeutic by engineering it to bind anotherprotein in the blood. In this manner, the drug attains its largemolecular size only after administration into the blood stream. Theaddition of affinity-matured serum albumin-binding peptides to antibodyfragments increased their circulation time 24 fold in mice (Dennis etal, 2002 J Bil Chem 277(38):35035). This method is complicated by thedynamics of albumin recycle by the neonatal Fc receptor (FcRn) and theuse of cysteine-constrained cyclic peptides for functionality.Alternatively, recombinant addition of large antibody fragments may bemade to a protein drug. This may cause structural as well asmanufacturing complications, e.g., because of the use of complex cyclicor large domains for functionality. Despite high affinity for albumin,they require the physical constraint of correctly forming a cyclicstructure prior to use. Methods of fusing larger antibody fragments maynot be amendable to proteins with an already complex folding structureor low expression yield.

The potential of chimeric antigen receptor T-cell therapies,antibody-coupled T-cell receptor (ACTR) therapies and other adoptiveT-cell therapies in effecting complete and durable responses has beendemonstrated in a number of malignant and infectious diseases. Thedevelopment of more potent T cells is limited, however, by safetyconcerns, highlighted by the occurrence of on-target and off-targettoxicities that, although uncommon, have been fatal on occasions. Timelypharmacological intervention can be effective in the management ofadverse events but adoptively transferred T cells can persist long term,along with any unwanted effects. T cells targeting differentiationantigens can be expected to also recognize nonmalignant cells thatexpress the same antigens, resulting in adverse events. For example,melanoma patients treated with T cells targeting melanocytedifferentiation antigens, such as MART-1 and gp100, often developvitiligo and uveitis. These on-target toxicities have been observedacross all forms of therapeutic approaches, including tumor-infiltratingcells, in vitro-expanded T-cell clones and TCR-transgenic cells. Ingeneral, on-target autoimmunity is associated with tumor regression andis more prominent in treatment approaches that are more efficacious.On-target but off-tumour toxicities can be immediately life-threatening.For example, patients with colorectal cancer with lung and livermetastases may develop respiratory distress within 15 min ofHER2-specific CAR T-cell infusion and may subsequently die frommultiorgan failure 5 days later. As T-cell therapy becomes moreeffective, acute toxicities have also become more evident. Cytokinerelease syndrome, which is characterized by fevers, rigors, hypotensionand hypoxia, has been observed in a number of CD19 CAR T-cell studies asa result of large-scale T-cell activation upon the recognition of CD19+malignant cells.

There is an ongoing need to provide therapeutic compositions through thecirculatory system that alleviate or prevent such diseases andconditions. There is a further a need for methods and compositions thatincrease the half-life, safety profile, and/or efficacy of suchtherapeutic compositions. Aspects of the invention address one or moreof the shortcomings of current methods and compositions.

SUMMARY OF THE INVENTION

In some aspects, disclosed herein is a method of reducing thecirculatory concentration of a target self-antibody. The methodcomprises the steps of administering to a human subject suffering fromor at risk of developing a self-antibody mediated disease, disorder orcondition, a pharmaceutical composition comprising a syntheticmembrane-receiver polypeptide complex, wherein the pharmaceuticalcomposition is administered in an amount effective to substantiallyreduce the circulatory concentration of the target self-antibody.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex has a volume of distribution equal to the plasma volume of thesubject.

In other embodiments, the synthetic membrane-receiver polypeptidecomplex has a volume of distribution of less than 0.09 l/kg.

In certain embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the self-antibody mediated disease, disorder or condition istreated, or a symptom thereof is decreased.

In other embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the self-antibody mediated disease, disorder or condition isprevented.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the targetself-antibody is substantially decreased during the treatment period.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the targetself-antibody is substantially decreased during the treatment periodsuch that one or more symptoms of the self-antibody mediated disease,disorder or condition is prevented, decreased or delayed.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the targetself-antibody is decreased at a rate greater than i) the endogenousclearance rate of the target self-antibody by the human subject, or ii)the endogenous production rate of the target self-antibody by the humansubject, or iii) both i) and ii).

In some embodiments, the circulatory concentration of the targetself-antibody is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% duringpart or the entirety of the treatment period.

In other embodiments, the circulatory concentration of the targetself-antibody is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% withinabout 1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23hours, or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks ofthe administration.

In some embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the targetself-antibody is substantially decreased for at least about one week,two weeks, three weeks, four weeks, one month, two months, three months,four months, five months, six months, or greater than six months.

In other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the targetself-antibody is substantially decreased for a period of time at leastas long as the treatment period.

In some embodiments, the treatment period is not longer than a year, sixmonths, three months, two months, one month, two weeks, one week, threedays, two days, one day.

In some embodiments, the time interval between administrations within atreatment period is no longer than the period in which the number ofsynthetic membrane-receiver polypeptide complexes in circulation isreduced to less than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number ofsynthetic membrane-receiver polypeptide complexes present in theadministered pharmaceutical composition.

In other embodiments, the frequency of administration is sufficient toeffectively reduce the circulatory concentration of the targetself-antibody below a level that is associated with a symptom of theself-antibody mediated disease, disorder or condition.

In some embodiments, the administering of the pharmaceutical compositionreduces the concentration of unbound target self-antibody or theconcentration of total target self-antibody in the circulatory system ofthe subject.

In some embodiments, the concentration of total target self-antibody isapproximately equal to the concentration of unbound and bound targetself-antibody in the circulatory system of the subject.

In certain embodiments, the pharmaceutical composition further comprisesa pharmaceutically active agent.

In certain embodiments, the method further comprises the step ofadministering a pharmaceutically active agent, wherein thepharmaceutically active agent is administered prior to, after, orconcurrent with the pharmaceutical composition.

In some embodiments, the pharmaceutical composition is administeredtopically or parenterally.

In some embodiments, the pharmaceutically active agent is selected froma biological agent, a small molecule agent, or a nucleic acid agent.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

In some embodiments, the method further comprises the step of selectingfor treatment a subject suffering from or at risk of a self-antibodymediated disease, disorder or condition selected from the groupconsisting of: type I diabetes, multiple sclerosis, ulcerative colitis,lupus, immune thrombocytopenia purpura, warm antibody hemolytic anemia,cold agglutinin disease, Goodpasture syndrome, antiphospholipid antibodysyndrome, and membranous glomerulonephritis.

In some embodiments, the synthetic membrane-receiver polypeptide complexis formulated for short-term duration in the circulatory system of thesubject.

In other embodiments, the synthetic membrane-receiver polypeptidecomplex is formulated for long-term duration in the circulatory systemof the subject.

In some embodiments, the receiver polypeptide is not substantiallydisassociated from the membrane in the circulatory system of thesubject.

In some embodiments, the receiver polypeptide is present in thecirculatory system for at least 21 days.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex comprises phosphatidylcholine, sphingomyelin,lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, or phosphatidic acid.

In some embodiments, the synthetic membrane-receiver polypeptide complexfurther comprises i) a CD47, CD55, or CD59 polypeptide or a functionalfragment thereof, or ii) a cell membrane polypeptide, or iii) both i)and ii).

In some embodiments, the synthetic membrane-receiver polypeptide complexcomprises a CD47, CD55, or CD59 polypeptide or a functional fragmentthereof in an amount effective for the polypeptide complex to reside inthe circulatory system for long-term duration.

In some embodiments, the synthetic membrane-receiver polypeptide complexdoes not contain a substantial amount of a replicating nucleic acid.

In some embodiments, the synthetic membrane-receiver polypeptide complexcomprises at least 10 copies, 100 copies, 1,000 copies, 10,000 copies,25,000 copies, 50,000 copies, or 100,000 copies of the receiverpolypeptide, and/or wherein the synthetic membrane-receiver polypeptidecomplex comprises a ratio of the receiver polypeptide relative to amembrane lipid selected from the group consisting ofphosphatidylcholine, sphingomyelin, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, orphosphatidic acid.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex comprises at least a second polypeptide in addition to thereceiver polypeptide.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex has catalytic activity for more than one substrate independentof the receiver polypeptide.

In some embodiments, the second polypeptide is associated with themembrane.

In certain embodiments, the receiver polypeptide is encoded by anexogenous nucleic acid.

In certain embodiments, the exogenous nucleic acid is not substantiallyretained by the synthetic membrane-receiver polypeptide complex.

In some embodiments, the expression of the receiver polypeptide iseffectively terminated.

In some embodiments, the receiver polypeptide is associated with themembrane.

In other embodiments, the receiver polypeptide is a fusion or a chimera.

In some embodiments, the fusion or chimera comprises at least one of anS domain, an A domain or a U domain, wherein the S domain is a surfacedomain exposed to the environment around the synthetic membrane-receiverpolypeptide complex, wherein the A domain is an anchor, wherein the Udomain faces the unexposed side of the synthetic membrane-receiverpolypeptide complex, and wherein the S domain, the A domain, and/or theU domain are of different polypeptide origin.

In some embodiments, the S domain and/or the A domain comprises at least6 or at least 30 amino acids.

In certain embodiments, the target self-antibody specifically recognizesglycoprotein (GP Ib-IX, IIb-IIIa, IV, or Ia-IIa), the NC1 domain ofcollagen α3 (IV), B2 glycoprotein-1, or phospholipase A2 receptor.

In certain embodiments, the receiver polypeptide comprises an antigenicpolypeptide selected from the group consisting of glycoprotein (GPIb-IX, IIb-IIIa, IV, or Ia-IIa), the NC1 domain of collagen α3 (IV), B2glycoprotein-1, or phospholipase A2 receptor, or an antigenic fragmentthereof.

In some embodiments, the S domain comprises the antigenic polypeptide orantigenic fragment thereof.

In some aspects, provided herein is a pharmaceutical compositionadministered by the methods disclosed herein.

In certain embodiments, the pharmaceutical composition further comprisesa pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition comprises apopulation of synthetic membrane-receiver polypeptide complexes.

In some embodiments, the pharmaceutical composition comprises at least1×10⁵ synthetic membrane-receiver polypeptide complexes. In certainembodiments, the synthetic membrane-receiver polypeptide complexes areprovided in a volume of about 10 nl, 100 nl, 1 μl, 10 μl, 100 μl, 1 ml,10 ml, 20 ml, or 50 ml.

In certain embodiments, the pharmaceutical composition comprises atleast 1×10¹¹ synthetic membrane-receiver polypeptide complexes. Incertain embodiments, the synthetic membrane-receiver polypeptidecomplexes are provided in a volume of about 1 ml, 10 ml, 20 ml, 50 ml,100 ml, 250 ml, or 500 ml.

In certain embodiments, the pharmaceutical composition is a compositionformulated for long-term storage.

In certain embodiments, the pharmaceutical composition is a compositionwhich is frozen.

In some embodiments, the pharmaceutical composition comprises apharmaceutically active agent.

In certain embodiments, the pharmaceutically active agent is selectedfrom a biological agent, a small molecule agent, or a nucleic acidagent.

In some aspects, provided herein is a dosage form comprising thecompositions disclosed herein formulated as a liquid suspension forintravenous injection.

In some aspects, provided herein is a medical device comprising acontainer holding the pharmaceutical compositions disclosed herein andan applicator for intravenous injection of the pharmaceuticalcomposition to the subject.

In some aspects, provided herein is a medical kit comprising thepharmaceutical compositions disclosed herein and a medical device forintravenous injection of the pharmaceutical composition to the subject.

In some aspects, provided herein is the synthetic membrane-receiverpolypeptide complex of the pharmaceutical composition administered bythe methods disclosed herein.

In some aspects, provided herein is a population of syntheticmembrane-receiver polypeptide complexes as disclosed herein.

In some embodiments, the population of synthetic membrane-receiverpolypeptide complexes are formulated as a liquid.

In other embodiments, the population of synthetic membrane-receiverpolypeptide complexes are frozen.

In some aspects, provided herein is an isolated receiver polypeptide ofthe synthetic membrane-receiver polypeptide complex as disclosed herein.

In some aspects, provided herein is an exogenous nucleic acid encodingthe receiver polypeptide disclosed herein.

In some aspects, provided herein is a synthetic membrane-receiverpolypeptide complex comprising: a receiver polypeptide capable ofinteracting with a target, and a membrane comprising a secondpolypeptide, wherein the synthetic membrane-receiver polypeptide complexhas catalytic activity independent of the receiver.

In some embodiments, the synthetic membrane-receiver polypeptide complexis formulated for intravenous administration to the circulatory systemof a mammalian subject, which for example can be a human.

In certain embodiments, the receiver polypeptide is capable of reducingthe concentration of unbound target or total target in the circulatorysystem of the subject.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex has a volume of distribution approximately equal or equivalentto the plasma volume of the subject.

In some embodiments, the synthetic membrane-receiver polypeptide complexhas a volume of distribution of less than 0.09 l/kg.

In some embodiments, the receiver polypeptide is present in thecirculatory system for substantially the duration of the syntheticmembrane-receiver polypeptide complex in the circulatory system of thesubject.

In some embodiments, the synthetic membrane-receiver polypeptide complexis formulated for short-term duration in the circulatory system of thesubject.

In some embodiments, the synthetic membrane-receiver polypeptide complexis formulated for long-term duration in the circulatory system of thesubject.

In certain embodiments, the receiver polypeptide is present in thecirculatory system for at least about 21 days.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex comprises phosphatidylcholine, sphingomyelin,lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, or phosphatidic acid.

In other embodiments, the synthetic membrane-receiver polypeptidecomplex further comprises a CD47, CD55, or CD59 polypeptide or afunctional fragment thereof.

In other embodiments, the synthetic membrane-receiver polypeptidecomplex comprises a CD47, CD55, or CD59 polypeptide or a functionalfragment thereof in an amount effective for the complex to reside in thecirculatory system for long-term duration.

In some embodiments, the interaction of the complex with a targetcomprises binding, degrading, cleaving and/or sequestering the target.

In other embodiments, the interaction of the complex with a targetcomprises altering an activity of the target.

In other embodiments, the interaction of the complex with a targetcomprises reducing an activity of the target.

In other embodiments, the interaction of the complex with a targetcomprises inactivating the target.

In some embodiments, the target is a self-antibody, a complementprotein, an immune complex, a serum amyloid protein, a metabolite or atoxin.

In other embodiments, the target is an inflammatory molecule, a cytokineor a chemokine.

In other embodiments, the target is a lipid or a carbohydrate, an aminoacid.

In other embodiments, the target is a virus, a viral antigen, anenvelope antigen or a capsid antigen.

In other embodiments, the target is a bacterium, a bacterial antigen, abacterial surface antigen, a secreted bacterial toxin, or a secretedbacterial antigen.

In other embodiments, the target is a fungus, a fungal antigen, a fungalcell surface antigen, a secreted fungal toxin, or a secreted fungalantigen.

In other embodiments, the target is DNA or RNA.

In other embodiments, the target is a circulating cell, an inflammatorycell, a tumor cell, or a metastatic cancer cell.

In certain embodiments, the receiver polypeptide is a complementreceptor 1 (CR1) polypeptide, a variant or functional fragment thereof.

In some embodiments, the CR1 polypeptide comprises one or more ShortConsensus Repeats (SCRs), Complement Control Proteins (CCPs) and/or LongHomologous Repeats (LHRs).

In certain embodiments, the receiver polypeptide is a duffy antigenreceptor complex (DARC), a variant or functional fragment thereof.

In other embodiments, the receiver polypeptide is an antibody, asingle-chain variable fragment, a nanobody, a diabody, or a DARPin.

In other embodiments, the receiver polypeptide is a lyase, a hydrolase,a protease, or a nuclease.

In other embodiments, the receiver polypeptide is exposed to theenvironment around the synthetic receiver polypeptide complex.

In other embodiments, the receiver polypeptide is located at theunexposed side of the synthetic receiver polypeptide complex.

In other embodiments, the receiver polypeptide is associated with themembrane.

In other embodiments, the receiver polypeptide is a fusion or a chimera.

In certain embodiments, the fusion or chimera comprises at least one ofan S domain, an A domain or a U domain, wherein the S domain is asurface domain exposed to the environment around the syntheticmembrane-receiver polypeptide complex, wherein the A domain is ananchor, wherein the U domain faces the unexposed side of the syntheticmembrane-receiver polypeptide complex, and wherein the S domain, the Adomain, and/or the U domain are of different polypeptide origin.

In some embodiments, the S domain and/or the A domain comprises at least6 or at least 30 amino acids.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex comprises at least 10 copies, 100 copies, 1,000 copies, 10,000copies, 25,000 copies, 50,000 copies, or 100,000 copies of the receiverpolypeptide, and/or wherein the synthetic membrane-receiver polypeptidecomplex comprises a ratio of the receiver polypeptide relative to amembrane lipid selected from the group consisting ofphosphatidylcholine, sphingomyelin, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, orphosphatidic acid.

In certain embodiments, the receiver polypeptide is encoded by arecombinant nucleic acid.

In certain embodiments, the recombinant nucleic acid is not retained bythe synthetic membrane-receiver polypeptide complex.

In certain embodiments, the expression of the receiver polypeptide iseffectively terminated.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex does not contain a substantial amount of a replicating nucleicacid.

In some aspects, provided herein is a pharmaceutical compositioncomprising a population of synthetic membrane-receiver polypeptidecomplexes as disclosed herein and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition comprises atleast 1×10⁵ synthetic membrane-receiver complexes.

In some embodiments, the synthetic membrane-receiver complexes areprovided in a volume of about 10 nl, 100 nl, 1 μl, 10 μl, 100 μl, 1 ml,10 ml, 20 ml, or 50 ml.

In certain embodiments, the pharmaceutical composition comprises atleast 1×10¹¹ synthetic membrane-receiver complexes.

In some embodiments, the synthetic membrane-receiver complexes areprovided in a volume of about 1 ml, 10 ml, 20 ml, 50 ml, 100 ml, 250 ml,or 500 ml.

In some embodiments, the pharmaceutical composition is a compositionformulated for long-term storage.

In some embodiments, the pharmaceutical composition is a compositionwhich is frozen.

In certain embodiments, the pharmaceutical composition comprises apharmaceutically active agent.

In some embodiments, the pharmaceutically active agent is selected froma biological agent, a small molecule agent, or a nucleic acid agent.

In some aspects, provided herein is a dosage form comprising thepharmaceutical compositions disclosed herein formulated as a liquidsuspension for intravenous injection.

In some aspects, provided herein is a medical device comprising acontainer holding the pharmaceutical composition disclosed herein and anapplicator for intravenous injection of the pharmaceutical compositionto a subject.

In some aspects, provided herein is a medical kit comprising thepharmaceutical composition disclosed herein and a medical device forintravenous injection of the pharmaceutical composition to a subject.

In some aspects, provided herein is a method of treating or preventing adisease, disorder or condition associated with the presence of or theconcentration of a target in the circulatory system of a mammaliansubject. The method comprises administering intravenously to the subjectthe pharmaceutical compositions disclosed herein in an amount effectiveto treat or prevent disease, disorder or condition.

In certain embodiments, the target is associated with the disease,disorder or condition.

In some aspects, provided herein is a method of modulating thecirculatory concentration of a target. The method comprisesadministering to a mammalian subject suffering from or at risk ofdeveloping a disease, disorder or condition associated with thepresence, absence, elevated or depressed concentration of the target inthe circulatory system of the subject, a pharmaceutical compositioncomprising a synthetic membrane-receiver polypeptide complex in anamount effective to substantially modulate the circulatory concentrationof the target.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex has a volume of distribution equal to the plasma volume of thesubject.

In certain embodiments, the administration is repeated when the amountof synthetic membrane-receiver polypeptide complexes in circulation isreduced to 50% of i) the concentration of the complexes that were firstadministered or ii) Cmax of the synthetic membrane-receiver polypeptidecomplexes in circulation.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex interacts with the target in circulation.

In certain embodiments, the interaction with the target comprisesbinding, degrading, cleaving and/or sequestering the target.

In other embodiments, the interaction with a target comprises alteringan activity of the target.

In other embodiments, the interaction with the target comprises reducingan activity of the target.

In other embodiments, the interaction with the target comprisesinactivating the target.

In other embodiments, the interaction with the target comprisescatalytically converting the target.

In certain embodiments, modulating consists of reducing the circulatoryconcentration of the target.

In certain embodiments, the presence or elevated level of the target inthe circulatory system of the subject is associated with the disease,disorder or condition.

In certain embodiments, the method further comprises increasing thecirculatory concentration of a non-target compound.

In certain embodiments, the absence or depressed level of the non-targetcompound in the circulatory system of the subject is associated with thedisease, disorder or condition.

In certain embodiments, the target is a biological compound, aninorganic compound, an organic compound, a gaseous compound or anelement.

In certain embodiments, the target is less than 1000 Da, less than 500Da, less than 250 Da, or less than 100 Da.

In certain embodiments, the target is more than 1 kDa.

In certain embodiments, the target is a polypeptide, a lipid, acarbohydrate, a nucleic acid, an amino acid, metabolite, or a smallmolecule.

In other embodiments, the target is an antibody, a complement factor, animmune complex, a serum amyloid protein, a bacterial pathogen, a fungalpathogen, a viral pathogen, or an infected, pathogenic, apoptotic,necrotic, aberrant or oncogenic mammalian cell.

In other embodiments, the target is an amyloid polypeptide.

In other embodiments, the target is a complement polypeptide.

In certain embodiments, the substantial modulation of the circulatoryconcentration of the target is reversible.

In other embodiments, the substantial modulation of the circulatoryconcentration of the target is temporally restricted.

In other embodiments, the substantial modulation of the circulatoryconcentration of the target is spatially restricted.

In some aspects, disclosed herein is a method of reducing thecirculatory concentration of a target serum amyloid protein. The methodcomprises the steps of administering to a mammalian subject sufferingfrom or at risk of developing an amyloidosis, a pharmaceuticalcomposition comprising a synthetic membrane-receiver complex, whereinthe pharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the target serumamyloid protein.

In certain embodiments, the synthetic membrane-receiver complex has avolume of distribution equal to the plasma volume of the subject. Insome embodiments, the synthetic membrane-receiver complex has a volumeof distribution of less than 0.09 l/kg.

In certain embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the amyloidosis is treated, or a symptom thereof is decreased.

In other embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the amyloidosis is prevented.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target serumamyloid protein is substantially decreased during the treatment period.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target serumamyloid protein is substantially decreased during the treatment periodsuch that one or more symptom of the amyloidosis is prevented, decreasedor delayed.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target serumamyloid protein is decreased at a rate greater than i) the endogenousclearance rate of the target serum amyloid protein by the mammaliansubject, or ii) the endogenous production rate of the target serumamyloid protein by the mammalian subject, or iii) both i) and ii).

In some embodiments, the circulatory concentration of the target serumamyloid protein is decreased by at least about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99%during part or the entirety of the treatment period.

In other embodiments, the circulatory concentration of the target serumamyloid protein is decreased by at least about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99%within about 1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or23 hours, or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeksof the administration.

In some embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target serumamyloid protein is substantially decreased for at least about one week,two weeks, three weeks, four weeks, one month, two months, three months,four months, five months, six months, or greater than six months.

In other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target serumamyloid protein is substantially decreased for a period of time at leastas long as the treatment period.

In some embodiments, the treatment period is not longer than a year, sixmonths, three months, two months, one month, two weeks, one week, threedays, two days, one day.

In some embodiments, the time interval between administrations within atreatment period is no longer than the period in which the number ofsynthetic membrane-receiver complexes in circulation is reduced to lessthan about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of syntheticmembrane-receiver complexes present in the administered pharmaceuticalcomposition.

In other embodiments, the frequency of administration is sufficient toeffectively reduce the circulatory concentration of the target serumamyloid protein below a level that is associated with a symptom of theamyloidosis.

In some embodiments, the administering of the pharmaceutical compositionreduces the concentration of unbound target serum amyloid protein or theconcentration of total target serum amyloid protein in the circulatorysystem of the subject.

In some embodiments, the concentration of total target serum amyloidprotein is approximately equal to the concentration of unbound and boundtarget serum amyloid protein in the circulatory system of the subject.

In some embodiments, the method further comprises the step of selectingfor treatment a subject suffering from or at risk of an amyloidosisselected from the group consisting of: A amyloidosis (AA), Ig lightchain amyloidosis (AL), transthyretin (TTR) amyloidosis, and fibrinogenamyloidosis.

In certain embodiments, the target serum amyloid protein is selectedfrom the group consisting of: amyloid P protein, amyloid A protein,light chain, misfolded transthyretin, and fibrinogen alpha chain.

In some embodiments, the receiver is associated with the membrane.Optionally, the receiver is a fusion or a chimera. If desired, thefusion or chimera may comprise at least one of an S domain, an A domainor a U domain, wherein the S domain is a surface domain exposed to theenvironment around the synthetic membrane-receiver complex, wherein theA domain is an anchor, wherein the U domain faces the unexposed side ofthe synthetic membrane-receiver complex, and wherein the S domain, the Adomain, and/or the U domain are of different polypeptide origin. In someembodiments, the S domain and/or the A domain comprise a polypeptidecomprising at least 6 or at least 30 amino acids. In some embodiments,the S domain comprises the antigenic polypeptide or antigenic fragmentthereof.

In some aspects, disclosed herein is a method of reducing thecirculatory concentration of a target immune complex. The methodcomprises the steps of administering to a mammalian subject sufferingfrom or at risk of developing an immune complex-associated disease,disorder or condition, a pharmaceutical composition comprising asynthetic membrane-complement receptor 1 (CR1) receiver complex, whereinthe pharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the target immunecomplex.

In certain embodiments, the synthetic membrane-CR1 receiver complex hasa volume of distribution equal to the plasma volume of the subject. Insome embodiments, the synthetic membrane-CR1 receiver complex has avolume of distribution of less than 0.09 l/kg.

In certain embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the immune complex-associated disease, disorder or condition istreated, or a symptom thereof is decreased.

In other embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the immune complex-associated disease, disorder or condition isprevented.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target immunecomplex is substantially decreased during the treatment period.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target immunecomplex is substantially decreased during the treatment period such thatone or more symptom of the a immune complex-associated disease, disorderor condition is prevented, decreased or delayed.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target immunecomplex is decreased at a rate greater than i) the endogenous clearancerate of the target immune complex by the mammalian subject, or ii) theendogenous production rate of the target immune complex by the mammaliansubject, or iii) both i) and ii).

In some embodiments, the circulatory concentration of the target immunecomplex is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% during partor the entirety of the treatment period.

In other embodiments, the circulatory concentration of the target immunecomplex is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% within about1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours,or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks of theadministration.

In some embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target immunecomplex is substantially decreased for at least about one week, twoweeks, three weeks, four weeks, one month, two months, three months,four months, five months, six months, or greater than six months.

In other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target immunecomplex is substantially decreased for a period of time at least as longas the treatment period.

In some embodiments, the treatment period is not longer than a year, sixmonths, three months, two months, one month, two weeks, one week, threedays, two days, one day.

In some embodiments, the time interval between administrations within atreatment period is no longer than the period in which the number ofsynthetic membrane-CR1 receiver complexes in circulation is reduced toless than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of syntheticmembrane—CR1 receiver complexes present in the administeredpharmaceutical composition.

In other embodiments, the frequency of administration is sufficient toeffectively reduce the circulatory concentration of the target immunecomplex below a level that is associated with a symptom of the immunecomplex-associated disease, disorder or condition.

In some embodiments, the administering of the pharmaceutical compositionreduces the concentration of unbound target immune complex or theconcentration of total target immune complex in the circulatory systemof the subject.

In some embodiments, the concentration of total target immune complex isapproximately equal to the concentration of unbound and bound targetimmune complex in the circulatory system of the subject.

In some embodiments, the method further comprises the step of selectingfor treatment a subject suffering from or at risk of a immunecomplex-associated disease, disorder or condition selected from thegroup consisting of: IgA nephropathy and lupus nephritis.

In certain embodiments, the target immune complex comprises i) IgM orIgG, and ii) C3b and/or C4b.

In some embodiments, the receiver is associated with the membrane.Optionally, the receiver is a fusion or a chimera. If desired, thefusion or chimera may comprise at least one of an S domain, an A domainor a U domain, wherein the S domain is a surface domain exposed to theenvironment around the synthetic membrane—CR1 receiver complex, whereinthe A domain is an anchor, wherein the U domain faces the unexposed sideof the synthetic membrane—CR1 receiver complex, and wherein the Sdomain, the A domain, and/or the U domain are of different polypeptideorigin. In some embodiments, the S domain and/or the A domain comprise apolypeptide comprising at least 6 or at least 30 amino acids. In someembodiments, the S domain comprises the antigenic polypeptide orantigenic fragment thereof.

In certain embodiments, the CR1 receiver polypeptide comprises one ormore of any one of a complement control protein (CCP) module, a shortconsensus repeat (SCR), and/or a long homologous repeat (LHRs)

In some aspects, disclosed herein is a method of reducing thecirculatory concentration of a target complement protein. The methodcomprises the steps of administering to a mammalian subject sufferingfrom or at risk of developing a disease, disorder or conditionassociated with the dysregulation of a complement protein, apharmaceutical composition comprising a synthetic membrane-receivercomplex, wherein the pharmaceutical composition is administered in anamount effective to substantially reduce the circulatory concentrationof the target complement protein.

In certain embodiments, the synthetic membrane-receiver complex has avolume of distribution equal to the plasma volume of the subject. Insome embodiments, the synthetic membrane-receiver complex has a volumeof distribution of less than 0.09 l/kg.

In certain embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the disease, disorder or condition associated with thedysregulation of a complement protein is treated, or a symptom thereofis decreased.

In other embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the disease, disorder or condition associated with thedysregulation of a complement protein is prevented.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target complementprotein is substantially decreased during the treatment period.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target complementprotein is substantially decreased during the treatment period such thatone or more symptom of the disease, disorder or condition associatedwith the dysregulation of a complement protein is prevented, decreasedor delayed.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target complementprotein is decreased at a rate greater than i) the endogenous clearancerate of the target complement protein by the mammalian subject, or ii)the endogenous production rate of the target complement protein by themammalian subject, or iii) both i) and ii).

In some embodiments, the circulatory concentration of the targetcomplement protein is decreased by at least about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99%during part or the entirety of the treatment period.

In other embodiments, the circulatory concentration of the targetcomplement protein is decreased by at least about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99%within about 1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or23 hours, or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeksof the administration.

In some embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target complementprotein is substantially decreased for at least about one week, twoweeks, three weeks, four weeks, one month, two months, three months,four months, five months, six months, or greater than six months.

In other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target complementprotein is substantially decreased for a period of time at least as longas the treatment period.

In some embodiments, the treatment period is not longer than a year, sixmonths, three months, two months, one month, two weeks, one week, threedays, two days, one day.

In some embodiments, the time interval between administrations within atreatment period is no longer than the period in which the number ofsynthetic membrane-receiver complexes in circulation is reduced to lessthan about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of syntheticmembrane-receiver complexes present in the administered pharmaceuticalcomposition.

In other embodiments, the frequency of administration is sufficient toeffectively reduce the circulatory concentration of the targetcomplement protein below a level that is associated with a symptom ofthe disease, disorder or condition associated with the dysregulation ofa complement protein.

In some embodiments, the administering of the pharmaceutical compositionreduces the concentration of unbound target complement protein or theconcentration of total target complement protein in the circulatorysystem of the subject.

In some embodiments, the concentration of total target complementprotein is approximately equal to the concentration of unbound and boundtarget complement protein in the circulatory system of the subject.

In some embodiments, the method further comprises the step of selectingfor treatment a subject suffering from or at risk of a disease, disorderor condition associated with the dysregulation of a complement proteinselected from the group consisting of: atypical hemolytic-uremicsyndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-relatedmacular degeneration, autoimmune hemolytic anemia, complement factor Ideficiency, and non-alcoholic steatohepatitis.

In certain embodiments, the target complement protein is selected fromthe group consisting of: C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, andC9.

In some aspects, disclosed herein is a method of modulating thecirculatory concentration of a target metabolite. The method comprisesthe steps of administering to a mammalian subject suffering from or atrisk of developing a metabolic disease, disorder or condition, apharmaceutical composition comprising a synthetic membrane-receivercomplex, wherein the pharmaceutical composition is administered in anamount effective to substantially modulate the circulatory concentrationof the target metabolite.

In certain embodiments, the synthetic membrane-receiver complex has avolume of distribution equal to the plasma volume of the subject. Insome embodiments, the synthetic membrane-receiver complex has a volumeof distribution of less than 0.09 l/kg.

In certain embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the metabolic disease, disorder or condition is treated, or asymptom thereof is decreased.

In other embodiments, the method comprises administering thepharmaceutical composition at least twice over a treatment period suchthat the metabolic disease, disorder or condition is prevented.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target metaboliteis substantially decreased during the treatment period.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of a metabolite issubstantially increased during the treatment period.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target metaboliteis substantially decreased during the treatment period such that one ormore symptom of the a metabolic disease, disorder or condition isprevented, decreased or delayed.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of a metabolite issubstantially increased during the treatment period such that one ormore symptom of the a metabolic disease, disorder or condition isprevented, decreased or delayed.

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of the target metaboliteis decreased at a rate greater than i) the endogenous clearance rate ofthe target metabolite by the mammalian subject, or ii) the endogenousproduction rate of the target metabolite by the mammalian subject, oriii) both i) and ii).

In yet other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times over a treatmentperiod such that the circulatory concentration of a metabolite isincreased at a rate greater than i) the endogenous clearance rate of ametabolite by the mammalian subject, or ii) the endogenous productionrate of a metabolite by the mammalian subject, or iii) both i) and ii).

In some embodiments, the circulatory concentration of the targetmetabolite is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% duringpart or the entirety of the treatment period.

In some embodiments, the circulatory concentration of a metabolite isincreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% during part or theentirety of the treatment period.

In other embodiments, the circulatory concentration of the targetmetabolite is decreased by at least about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% withinabout 1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23hours, or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks ofthe administration.

In other embodiments, the circulatory concentration of a metabolite isincreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% within about 1, 5, 10,15, 20, 30, 40, or 50 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or 1, 2, 3,4, 5, or 6 days or about 1, 2, 3, 4, 5, or 6 weeks of theadministration.

In some embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target metaboliteis substantially decreased for at least about one week, two weeks, threeweeks, four weeks, one month, two months, three months, four months,five months, six months, or greater than six months.

In some embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of a metabolite issubstantially increased for at least about one week, two weeks, threeweeks, four weeks, one month, two months, three months, four months,five months, six months, or greater than six months.

In other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of the target metaboliteis substantially decreased for a period of time at least as long as thetreatment period.

In other embodiments, the method comprises administering thepharmaceutical composition a sufficient number of times a treatmentperiod such that the circulatory concentration of a metabolite issubstantially increased for a period of time at least as long as thetreatment period.

In some embodiments, the treatment period is not longer than a year, sixmonths, three months, two months, one month, two weeks, one week, threedays, two days, one day.

In some embodiments, the time interval between administrations within atreatment period is no longer than the period in which the number ofsynthetic membrane-receiver complexes in circulation is reduced to lessthan about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of syntheticmembrane-receiver complexes present in the administered pharmaceuticalcomposition.

In other embodiments, the frequency of administration is sufficient toeffectively reduce the circulatory concentration of the targetmetabolite below a level that is associated with a symptom of themetabolic disease, disorder or condition.

In other embodiments, the frequency of administration is sufficient toeffectively increase the circulatory concentration of a metabolite abovea level that is associated with a symptom of the metabolic disease,disorder or condition.

In some embodiments, the administering of the pharmaceutical compositionreduces the concentration of unbound target metabolite or theconcentration of total target metabolite in the circulatory system ofthe subject.

In some embodiments, the concentration of total target metabolite isapproximately equal to the concentration of unbound and bound targetmetabolite in the circulatory system of the subject.

In some embodiments, the administering of the pharmaceutical compositionincreases the concentration of an unbound metabolite or theconcentration of total metabolite in the circulatory system of thesubject.

In some embodiments, the method further comprises the step of selectingfor treatment a subject suffering from or at risk of a metabolicdisease, disorder or condition selected from the group consisting of:Phenylketonuria (PKU), Adenosine Deaminase Deficiency-Severe CombinedImmunodeficiency (ADA-SCID), Mitochondrial NeurogastrointestinalEncephalopathy (MNGIE), Primary Hyperoxaluria, Alkaptonuria, andThrombotic Thrombocytopenic Purpura (TTP).

In certain embodiments, the target metabolite is selected from the groupconsisting of: Phenylalanine, Adenosine, Thymidine, Deoxyuridine,Oxalate, Homogentisate, von Willenbrand Factor.

In some embodiments, the receiver is associated with the membrane.Optionally, the receiver is a fusion or a chimera. If desired, thefusion or chimera may comprise at least one of an S domain, an A domainor a U domain, wherein the S domain is a surface domain exposed to theenvironment around the synthetic membrane-receiver complex, wherein theA domain is an anchor, wherein the U domain faces the unexposed side ofthe synthetic membrane-receiver complex, and wherein the S domain, the Adomain, and/or the U domain are of different polypeptide origin. In someembodiments, the S domain and/or the A domain comprise a polypeptidecomprising at least 6 or at least 30 amino acids. In some embodiments,the S domain comprises the antigenic polypeptide or antigenic fragmentthereof.

In certain embodiments, the receiver polypeptide is selected from thegroup consisting of: Phenylalanine Hydroxylase, Adenosine Deaminase,Thymidine Phosphorylase, Glyoxalate Reductase, Homogentisate Reductase,ADAMTS13.

Aspects of the invention relate to synthetic membrane receiver complexesthat comprise non-polypeptide receivers, such as nucleic acids, lipids,carbohydrates and/or small molecules. In some embodiments, the receiveris associated with the membrane. Optionally, the receiver is a fusion ora chimera with a polypeptide. If desired, the fusion or chimera maycomprise at least one of an S domain, an A domain or a U domain, whereinthe S domain is a surface domain exposed to the environment around thesynthetic membrane-receiver complex, wherein the A domain is an anchor,wherein the U domain faces the unexposed side of the syntheticmembrane-receiver complex, and wherein the S domain, the A domain,and/or the U domain are of different origin. In some embodiments, the Sdomain and/or the A domain comprise a polypeptide comprising at least 6or at least 30 amino acids. In some embodiments, the S domain comprisesthe antigenic polypeptide or antigenic fragment thereof.

In certain embodiments, the pharmaceutical compositions described hereincomprise a population of synthetic membrane-receiver complexes such asat least 1×10⁵ synthetic membrane-receiver complexes, optionally in avolume of about 10 nl, 100 nl, 1 μl, 10 μl, 100 μl, 1 ml, 10 ml, 20 ml,or 50 ml. In certain embodiments, the pharmaceutical compositionsdescribed herein comprise a population of synthetic membrane-receivercomplexes such as at least 1×10¹¹ synthetic membrane-receiver complexes,optionally in a volume of about 1 ml, 10 ml, 20 ml, 50 ml, 100 ml, 250ml, or 500 ml.

In some aspects, provided herein is the synthetic membrane-receivercomplex of the pharmaceutical composition administered by the methodsdisclosed herein.

In some aspects, provided herein is a population of syntheticmembrane-receiver complexes as disclosed herein. Optionally, thepopulation of synthetic membrane-receiver complexes is formulated as aliquid. Alternatively, the population of synthetic membrane-receivercomplexes is frozen.

In some aspects, provided herein is an isolated receiver of thesynthetic membrane-receiver complex as disclosed herein.

In some aspects, provided herein is an exogenous nucleic acid encodingthe receiver disclosed herein.

In some aspects, provided herein is a synthetic membrane-receivercomplex comprising: a receiver capable of interacting with a target, anda membrane comprising a polypeptide that is not the receiver, whereinthe synthetic membrane-receiver complex has catalytic activityindependent of the receiver.

In some embodiments, the synthetic membrane-receiver complex comprises areceiver that is not a polypeptide.

In some embodiments, any synthetic membrane-receiver complex describedherein, including those comprising a polypeptide receiver, optionallycomprise a payload, such as a therapeutic agent.

Aspects of the invention relate to isolated, enucleated erythroid cellcomprising a receiver polypeptide that is functionally active when theenucleated erythroid cell is administered to the circulatory system of asubject. In some embodiments, the erythroid cell is a human cell.

Aspects of the invention relate to isolated, functional erythroidprecursor cell comprising a receiver polypeptide that is encoded by anexogenous nucleic acid, wherein the expression of the receiverpolypeptide does not substantially alter: the expression of a surfacemarker, selected from the group consisting of GPA, cKit, and TR when thefunctional erythroid precursor cell differentiates; the rate ofenucleation when the functional erythroid precursor cell terminallymatures; and/or the rate of expansion when the functional erythroidprecursor cell expands in culture, wherein the alteration is compared toan isolated, uncultured erythroid precursor cell of the same stage andlineage not comprising the polypeptide receiver.

Aspects of the invention relate to isolated erythroid cell populationscomprising a plurality of functional erythroid cells comprising areceiver polypeptide localized to an exterior surface of the erythroidcells, wherein the population is substantially free of non-erythroidcells. In some embodiments, the population comprises greater than 5-95%of enucleated erythroid cells.

Aspects of the invention relate to isolated erythroid cell populationscomprising a plurality of functional erythroid cells comprising areceiver polypeptide encoded by an exogenous nucleic acid, whereinduring enucleation the receiver polypeptide is retained by the erythroidcell whereas the exogenous nucleic acid is not retained. In someembodiments, the population comprises greater than 5-95% of enucleatederythroid cells, optionally in the absence of: i) an enrichment stepand/or ii) co-culturing with non-erythroid cells.

Aspects of the invention relate to isolated erythroid cell populationscomprising a plurality of functional erythroid cells comprising areceiver polypeptide encoded by an exogenous nucleic acid, whereinduring enucleation the receiver polypeptide is retained by the erythroidcell whereas the exogenous nucleic acid is not retained, and wherein theresulting functional enucleated erythroid cell exhibits substantiallythe same osmotic membrane fragility as an isolated, uncultured erythroidcell not comprising the polypeptide receiver.

Aspects of the invention relate to isolated erythroid cell populationscomprising a plurality of functional erythroid precursor cells insubstantially the same stage of differentiation and/or cell cycle stage,wherein the precursor cells comprise an exogenous nucleic acid encodinga receiver polypeptide, and wherein a majority of erythrocyte precursorcells is capable of differentiating into mature erythrocytes that retainthe receiver polypeptide without retaining the exogenous nucleic acid.

Aspects of the invention relate to isolated erythroid cell populationscomprising a plurality of functional erythroid cells comprising areceiver polypeptide, wherein an exogenous nucleic acid encoding thereceiver polypeptide is introduced into a cultured or freshly isolatederythroid cell precursor and wherein after introduction of the exogenousnucleic acid the functional erythroid cells expand from the precursorcells by more than 20,000-fold in culture. In some embodiments, thepopulation comprises greater than 5-95% of enucleated erythroid cells,optionally in the absence of: i) an enrichment step and/or ii)co-culturing with non-erythroid cells.

Aspects of the invention relate to an isolated erythroid cell populationthat is cultured from a functional erythrocyte precursor cell comprisingan exogenous nucleic acid, the population comprising: a pyrenocyte, afunctional nucleated erythroid cell and a functional enucleatederythroid cell, wherein the functional nucleated erythroid cell and thefunctional enucleated erythroid cell comprise an receiver polypeptideencoded by the exogenous nucleic acid, and wherein the receiverpolypeptide is retained by the functional enucleated erythroid cell,whereas the exogenous nucleic acid is not retained by the enucleatederythroid cell. In some embodiments, the enucleated, functionalerythroid cell exhibits substantially the same osmotic membranefragility as an isolated, uncultured erythroid cell not comprising thepolypeptide receiver.

In some embodiments, the erythroid cell populations described hereincomprise greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or greater than10% fetal hemoglobin.

In some embodiments, the functional erythroid cell exhibits at least 10copies, 100 copies, 1,000 copies, 10,000 copies, 25,000 copies, 50,000copies, or 100,000 copies of the receiver polypeptide per cell.

In certain embodiments, a plurality of functional erythroid cells losesa substantial portion of its cell membrane after being administered tothe circulatory system of a subject.

In certain embodiments, the functional erythroid cells comprise areceiver polypeptide that interacts with a target. In some embodiments,interacting with a target comprises binding to the target, degrading thetarget, cleaving the target, and/or sequestering the target.

In some embodiments, the receiver polypeptide is displayed on the cellsurface. In other embodiments, the receiver polypeptide is localized inthe interior of the functional erythroid cell.

In certain embodiments, the functional erythroid cells comprise areceiver polypeptide that is selected from the group consisting of: anantibody, a single-chain variable fragment, a nanobody, a diabody, adarbin, a lyase, a hydrolase, a protease, a nuclease, and a DNase.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that interacts with a target that is selected from the groupconsisting of: an immune complex, an inflammatory molecule, aninflammatory cell, a lipid, a carbohydrate, an amino acid, a virus, abacterium, a bacterial toxin, a fungus, a fungal toxin, a DNA, an RNA, acell, a circulating cell, a tumor cell, a metastatic cancer cell, ametabolite, a plant toxin, a cytokine, a chemokine, a complement cascadefactor, and a clotting cascade factor.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is fused to an endogenous polypeptide. In certainembodiments, the endogenous polypeptide is an intracellular polypeptide.In some embodiments, the endogenous polypeptide is an extracellularpolypeptide. In some embodiments, the endogenous polypeptide ismembrane-bound.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is fused to an endogenous extracellular polypeptide.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that conjugated to the erythroid cell.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that interacts with the target intercellularly.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is localized in the cytosol of the erythroid cell.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is located in the cell membrane of the erythroid cell.

In certain embodiments, the functional erythroid cells comprise aplurality of receiver polypeptides. In some embodiments, a firstreceiver polypeptide is located in the cytosol of the functionalerythroid cell and a second receiver polypeptide is located on the cellsurface of the functional erythroid cell.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is an Fv portion of an antibody that binds a botulinumtoxin and the target is a botulinum toxin.

In other embodiments, the functional erythroid cells comprise a receiverpolypeptide that is a complement receptor 1 and the target is acirculating immune complex.

In yet other embodiments, the functional erythroid cells comprise areceiver polypeptide that is a duffy antigen receptor complex (DARC) andthe target is a circulating chemokine.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is phenylalanine hydroxylase (PAH) and the target isphenylalanine.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is expressed as a fusion of the C-terminus of acytoplasmic beta globin protein.

In other embodiments, the functional erythroid cells comprise a receiverpolypeptide that is an exonuclease and wherein the target is acirculating cell-free DNA molecule.

In yet other embodiments, the functional erythroid cells comprise areceiver polypeptide that is expressed as a fusion of the N-terminus ofendogenous glycophorin A.

In some embodiments, the functional erythroid cells comprise a receiverpolypeptide that is attached extracellularly on the erythroid cell bycovalent bond formation. In some embodiments, the covalent bond isformed by an isopeptidase. In some embodiments, the isopeptidase isSpyTag/SpyCatcher. In some embodiments, the SpyTag is expressed on thesurface of the cell. In some embodiments, the SpyTag is fused to anextracellular terminus of a transmembrane protein. In some embodiments,the SpyTag is an in-frame fusion in an extracellular region of amulti-pass membrane protein. In some embodiments, the SpyTag is fused toa GPI-linked protein. In some embodiments, the SpyCatcher is fused tothe receiver polypeptide. In some embodiments, the receiver polypeptidefused to SpyCatcher is expressed and/or secreted in the same functionalerythroid cell that expresses the SpyTag fusion. In some embodiments,the receiver polypeptide fused to SpyCatcher is expressed by anexogenous protein production system and then contacted with thefunctional erythroid cell that expresses the SpyTag fusion. In someembodiments, the SpyTag is replaced with SpyCatcher and the SpyCatcheris replaced with SpyTag. In some embodiments, the receiver polypeptideis anchored intracellularly in the functional erythroid cell by covalentbond formation. In some embodiments, the covalent bond is formed by anisopeptidase. In some embodiments, the isopeptidase isSpyTag/SpyCatcher. In some embodiments, the SpyTag is expressed in theintracellular space of the cell. In some embodiments, the SpyTag isfused to an intracellular terminus of a membrane protein. In someembodiments, the SpyTag is an in-frame fusion in an intracellular regionof a multi-pass membrane protein. In some embodiments, the SpyTag isfused to an endogenous intracellular protein. In some embodiments, theSpyTag is fused to a cytoskeletal protein. In some embodiments, theSpyCatcher is fused to the receiver polypeptide. In some embodiments,the receiver polypeptide fused to SpyCatcher is expressed in theintracellular space of the same functional erythroid cell that expressesthe SpyTag fusion. In some embodiments, the SpyTag is replaced withSpyCatcher and the SpyCatcher is replaced with SpyTag.

Aspects of the invention relate to methods of generating functionalerythroid cells comprising a receiver polypeptide, the methodscomprising contacting an erythroid cell with a receiver and exposing theerythroid cell to a controlled cell injury. In certain embodiments, thecontrolled cell injury is cell deformation, electroporation,sonoporation, liposomal transfection, or salt-based transfection. Insome embodiments, the cell is contacted with an mRNA that encodes thereceiver polypeptide. In some embodiments, the contacting results in anuptake and translation of the mRNA encoding the receiver polypeptide bythe erythroid cell or erythriod cell precursor.

In certain embodiments, the populations of erythroid cells describedherein are maintained and/or propagated in vitro. In other embodiments,the populations of erythroid cells described herein are lyophilized. Inyet other embodiments, the populations of erythroid cells describedherein are frozen.

Aspects of the invention relate to methods of contacting a targetcomprising: introducing into a biological sample or a subject theerythroid cell populations described herein, and maintaining the contactof the erythroid cell population with the sample or subject for a timesufficient for a functional erythroid cell from the population tointeract with a target in the sample or subject. In some embodiments,interacting with a target comprises binding to the target, degrading thetarget, cleaving the target, and/or sequestering the target. In certainembodiments, the methods of contacting a target are carried out invitro. In other embodiments, the methods of contacting a target arecarried out in vivo, e.g. in an animal. In some embodiments, the methodsof contacting a target further comprise contacting the target with anassayable moiety. In some embodiments, the assayable moiety is used todetermine the rate and/or degree of interaction between the functionalerythroid cell and the target.

Aspects of the invention relate to pharmaceutical compositionscomprising the erythroid cell populations comprising the functionalerythroid cells comprising a receiver described herein. Optionally, thepharmaceutical compositions comprising the erythroid cell populationsfurther comprise a pharmaceutically acceptable carrier. Optionally thethe pharmaceutical compositions comprising the erythroid cellpopulations further comprise a therapeutic agent.

Aspects of the invention relate to methods of treating, preventing, ormanaging a disease or condition, comprising administering to a subjectin need of such treatment, prevention or management, a therapeuticallyor prophylactically effective amount of the pharmaceutical compositioncomprising a population of functional erythroid cells comprising areceiver, thereby treating, preventing, or managing the disease orcondition.

Aspects of the invention relate to pharmaceutical compositionscomprising a population of functional erythroid cells comprising areceiver for use in any of the methods of treatment or preventiondescribed herein. In some embodiments, the receiver polypeptideinteracts with a target residing in the circulatory system of thesubject. In some embodiments, the presence, absence, elevated ordepressed level of the target is associated with a disease, disorder orcondition. In some embodiments, interacting with a target comprisesbinding to the target, degrading the target, cleaving the target, and/orsequestering the target. In some embodiments, the administration of thepharmaceutical compositions comprising a population of functionalerythroid cells comprising a receiver results in a substantial reductionof the concentration or number of the target in the circulatory systemof the subject.

Aspects of the invention relate to pharmaceutical compositionscomprising a plurality of functional erythroid cells comprising areceiver polypeptide, wherein the erythroid cells exhibit the receiverpolypeptide in or on the cell, and wherein the receiver polypeptide whenthe functional erythroid cell is administered to the circulatory systemof a subject: does not substantially affect the circulation clearancetime of the functional erythroid cell when compared to a unmodifiederythroid cell in a control animal, and/or does not activate fibrinogenbreakdown, measured by circulating levels of fibrinopeptide A and/orfibrinopeptide B, compared to an unmodified erythroid cell.

Aspects of the invention relate to methods for culturing the functionalerythroid cell population of described herein, comprising using one ormore culturing factors selected from the group consisting of stem cellfactor, IL-3, IL-6, insulin, transferrin, erythropoietin,hydrocortisone, and estrogens to culture the functional erythroid cells.

Aspects of the invention relate to populations of at least 10¹⁰ cellscomprising at least 10% reticulocytes of the same blood group, wherein aplurality of the reticulocytes comprises a receiver polypeptide.

Aspects of the invention relate to a pharmaceutical compositioncomprising a synthetic membrane-receiver polypeptide complex for use inthe treatment of any of the diseases, disorders, or conditions disclosedherein.

Aspects of the invention relate to a pharmaceutical compositioncomprising a synthetic membrane-receiver polypeptide complex for use inthe treatment of a disease, disorder, or condition associated with thepresence of or the concentration of a target in the circulatory systemof a mammalian subject.

Aspects of the invention relate to a pharmaceutical compositioncomprising a synthetic membrane-receiver polypeptide complex for use inthe modulation of the circulatory concentration of a target.

BRIEF DESCRIPTION OF THE FIGURES

The figures are meant to be illustrative of one or more features,aspects, or embodiments of the invention and are not intended to belimiting.

FIG. 1A-FIG. 1D is a collection of flow cytometry plots of red bloodcells contacted with fluorescently labeled IgG encapsulated withinliposomes. Cells are shown that are incubated with no liposomes (FIG.1A, FIG. 1D), a low dose of liposomes (FIG. 1B, FIG. 1E), and a highdose of liposomes (FIG. 1C, FIG. 1F). On the bottom histograms, thepercentage of cells that are fluorescent is shown.

FIG. 2 is a plot of cell surface expression levels assessed byquantitative flow cytometry. The plot shows of various cell surfacereceptors—glycophorin A (solid triangles), cKIT (dashed squares),transferrin receptor (dotted diamonds)—and an exogenous surfacetransgene (open circles) during the course of erythroid celldifferentiation.

FIG. 3A-FIG. 3AP is a collection of flow cytometry plots and Westernblots that demonstrate the expression of a vast array of exemplaryreceivers on the surface, in the cytoplasm, as fusions, and as intactproteins, in three cell types, enucleated erythroid cells, nucleatederythroid precursor cells, and erythroleukemic cells.

FIG. 3A-FIG. 3N shows the exogenous expression of surface andcytoplasmic proteins on enucleated cultured erythroid cells.

FIG. 3A—Expression of glycophorin A with an HA epitope tag at thecytoplasmic C terminus assessed by expression of co-translated GFP.

FIG. 3B—Expression of glycophorin A with an HA epitope tag at the Nterminus between the leader sequence and the body of the gene assessedby anti-HA staining.

FIG. 3C—Expression of complement receptor 1-derived fragment of ˜70 kDawith an HA epitope tag at the N terminus assessed by anti-HA staining.

FIG. 3D—Expression of antibody scFv as N terminal fusion to glycophorinA assessed by anti-HA staining.

FIG. 3E—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 71 amino acids assessed by anti-HA staining.

FIG. 3F—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 79 amino acids assessed by anti-HA staining.

FIG. 3G—Expression of CD55 with HA epitope tag at the extracellular Nterminus after the leader sequence assessed by anti-HA staining.

FIG. 3H—Expression of CD59 with HA epitope tag at the extracellular Nterminus after the leader sequences assessed by anti-HA staining.

FIG. 3I—Expression of antibody scFv fused to N-terminus of CD55-derivedfragment of 37 amino acids, assessed by anti-HA Western blot.

FIG. 3J—Cytoplasmic expression of adenosine deaminase fused to HA tagassessed by anti-HA Western blot. Expected size approximately 40 kDa.

FIG. 3K—Cytoplasmic expression of phenylalanine hydroxylase fused to HAtag assessed by anti-HA Western blot. Expected size approximately 33kDa.

FIG. 3L—Cytoplasmic expression of phenylalanine hydroxylase fused toadenosine deaminase and an HA tag assessed by anti-HA Western blot.

FIG. 3M—Cytoplasmic expression of adenosine deaminase fused to theintracellular C terminus of glycophorin A assessed by anti-HA Westernblot. Expected size approximately 55 kDa.

FIG. 3N—Cytoplasmic expression of phenylalanine hydroxylase fused to theintracellular C terminus of glycophorin A assessed by anti-HA Westernblot. Expected size approximately 50 kDa.

FIG. 3O-FIG. 3AJ shows the exogenous expression of surface andcytoplasmic proteins on nucleated cultured erythroid precursor cells.

FIG. 3O—Expression of glycophorin A with an HA epitope tag at thecytoplasmic C terminus assessed by expression of co-translated GFP.

FIG. 3P—Expression of glycophorin A with an HA epitope tag at the Nterminus between the leader sequence and the body of the gene assessedby anti-HA staining.

FIG. 3Q—Overexpression of complement receptor 1 assessed by anti-CR1staining.

FIG. 3R—Expression of complement receptor 1-derived fragment of ˜70 kDawith an HA epitope tag at the N terminus assessed by anti-HA staining.

FIG. 3S—Expression of complement receptor 1-derived fragment of ˜210 kDawith an HA epitope tag at the N terminus assessed by anti-HA staining.

FIG. 3T—Expression of complement receptor 1-derived fragment of ˜230 kDafused to the N terminus of glycophorin A with an HA epitope tag at the Nterminus assessed by anti-HA staining.

FIG. 3U—Expression of antibody scFv as N terminal fusion to glycophorinA assessed by anti-HA staining.

FIG. 3V—Expression of antibody scFv fused to the extracellular Cterminus of Kell, assessed by anti-HA staining. Expected sizeapproximately 108 kDa.

FIG. 3W—Expression of HA tag fused to the extracellular C terminus ofKell, assessed by anti-HA staining.

FIG. 3X—Expression of Kell-derived fragment of 71 amino acids with HAtag at the C (extracellular) terminus assessed by anti-HA staining.

FIG. 3Y—Expression of Kell-derived fragment of 79 amino acids with HAtag at the C terminus assessed by anti-HA staining.

FIG. 3Z—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 71 amino acids assessed by anti-HA staining.

FIG. 3AA—Expression of antibody scFv fused to C terminus of Kell-derivedfragment of 79 amino acids assessed by anti-HA staining.

FIG. 3AB—Expression of CD55 with HA epitope tag at the extracellular Nterminus after the leader sequence assessed by anti-HA staining.

FIG. 3AC—Expression of CD59 with HA epitope tag at the extracellular Nterminus after the leader sequences assessed by anti-HA staining.

FIG. 3AD—Expression of antibody scFv fused to N-terminus of CD55-derivedfragment of 37 amino acids, assessed by anti-HA staining.

FIG. 3AE—Expression of antibody scFv fused to N-terminus of CD59assessed by anti-HA staining.

FIG. 3AF—Cytoplasmic expression of adenosine deaminase fused to HA tagassessed by anti-HA Western blot. Expected size approximately 40 kDa.

FIG. 3AG—Cytoplasmic expression of phenylalanine hydroxylase fused to HAtag assessed by anti-HA Western blot. Expected size approximately 33kDa.

FIG. 3AH—Cytoplasmic expression of phenylalanine hydroxylase fused toadenosine deaminase and an HA tag assessed by flow cytometry forfluorescence from co-translated GFP.

FIG. 3AI—Cytoplasmic expression of adenosine deaminase fused to theintracellular C terminus of glycophorin A assessed by anti-HA Westernblot. Expected size approximately 55 kDa.

FIG. 3AJ—Cytoplasmic expression of phenylalanine hydroxylase fused tothe intracellular C terminus of glycophorin A assessed by anti-HAWestern blot. Expected size approximately 50 kDa.

FIG. 3AK-FIG. 3AP shows the exogenous expression of surface andcytoplasmic proteins on K562 erythroleukemia cells.

FIG. 3AK—Overexpression of complement receptor 1 assessed by anti-CR1staining.

FIG. 3AL—Expression of antibody scFv as N terminal fusion to glycophorinA assessed by anti-HA staining.

FIG. 3AM—Expression of antibody scFv fused to N-terminus of CD55-derivedfragment of 37 amino acids, assessed by anti-HA staining.

FIG. 3AN—Expression of antibody scFv fused to N-terminus of CD59assessed by anti-HA staining.

FIG. 3AO—Cytoplasmic expression of adenosine deaminase fused to HA tagassessed by anti-HA Western blot. Expected size approximately 40 kDa.

FIG. 3AP—Cytoplasmic expression of phenylalanine hydroxylase fused to HAtag assessed by anti-HA Western blot. Expected size approximately 33kDa.

FIG. 4A-FIG. 4C is a collection of flow cytometry histograms thatmeasure fluorescence in primary platelets that have been transfectedwith mRNA encoding a fluorescent protein (GFP). (FIG. 4A) Untransfectedplatelets. (FIG. 4B) Platelets transfected with 3 ug GFP mRNA. (FIG. 4C)Platelets transfected with 6.8 ug GFP mRNA.

FIG. 5A-FIG. 5C shows protein expression and enzymatic activity oftransgenic erythroid cells in culture. (FIG. 5A) is a Western blot ofexogenously expressed adenosine deaminase detected with an anti-HAantibody over the course of differentiation, from nucleated precursorcells (“Diff I D5”) through to enucleated erythroid cells (“Diff IIID8”). (FIG. 5B) is a bar chart of inosine produced from adenosine byintact adenosine deaminase-expressing 293T cells. (FIG. 5C) is a Westernblot of the exogenously expressed phenylalanine hydroxylase detectedwith an anti-HA antibody at various time points over the course ofdifferentiation, from nucleated precursor cells (“Diff I D5”) through toenucleated erythroid cells (“Diff III D8”). (FIG. 5D) is a bar chart oftyrosine produced from phenylalanine by lysates of culturedphenylalanine hydroxylase-expressing enucleated erythroid cells.

FIG. 6A-FIG. 6B shows immune complex capture and transfer to macrophagesby cultured erythroid cells that overexpress complement receptor 1(CR1). (FIG. 6A) is a flow cytometry plot that shows the capture offluorescent immune complexes (white histogram) and complement-deficientimmune complexes (shaded histogram) by cultured erythroid cells thatoverexpress CR1. (FIG. 6B) is a bar chart of flow cytometry dataassessing the uptake of fluorescent immune complexes (hashed bars),complement deficient immune complexes (gray bars), or no immunecomplexes (black bars) by macrophages (left set) or macrophagesincubated with cultured erythroid cells that overexpress CR1 (rightset).

FIG. 7A-FIG. 7D shows the activity of an antibody scFv that bindshepatitis B surface antigen (scFv) on the surface of a culturederythroid cell. (FIG. 7A) is a flow cytometry histogram showing bindingof 450 nM antigen (white histogram) or no antigen (gray histogram).(FIG. 7B) is a titration of binding signal assessed by flow cytometryfor a range of antigen concentrations. (FIG. 7C-FIG. 7D) are flowcytometry plots of blood cells from mice that had been injected withfluorescent antigen and cultured erythroid cells that (FIG. 7C) do notor (FIG. 7D) do express scFv. The y-axis measures antigen fluorescence.The x-axis measures fluorescence of the cultured cells.

FIG. 8A-FIG. 8D shows the specific clearance of circulating antibodiesmediated by membrane-receiver complexes in vivo. (FIG. 8A) is a set offlow cytometry plots that show no binding (left) and binding (right) ofcirculating Dylight650-labeled mouse anti-HA antibody to CFSE-labeledcultured human erythroid cells isolated from a recipient mouse thateither do not (left) or do (right) express HA epitope tag on theirsurface. The x-axis measures CFSE fluorescence. The y-axis measuresanti-HA antibody Dylight650 fluorescence. (FIG. 8B) is data from an HAepitope tag substrate ELISA comparing anti-HA antibody levels over timein plasma collected from mice injected with anti-HA antibody (opencircles, solid line), anti-HA antibody followed by cultured humanerythroid cells that do not express HA epitope tag (dashed line), oranti-HA antibody followed by cultured human erythroid cells that doexpress HA epitope tag (dotted line). (FIG. 8C) is a set of flowcytometry plots that show no binding (left) and binding (right) ofDylight650-labeled mouse anti-biotin antibody to CFSE-labeled primaryhuman erythrocytes that either are not (left) or are (right) conjugatedwith biotin on their surface. The x-axis measures CFSE fluorescence. They-axis measures anti-biotin antibody Dylight650 fluorescence. (FIG. 8D)is data from a biotin substrate ELISA comparing anti-biotin antibodylevels over time in plasma collected from mice injected with anti-biotinantibody (open circles, solid line), anti-biotin antibody followed bycultured human erythroid cells that are not conjugated to biotin (dashedline), or anti-biotin antibody followed by cultured human erythroidcells that are conjugated to biotin (dotted line).

FIG. 9A-FIG. 9B shows the clearance rate of cultured human eyrthroidcells in a mouse. (FIG. 9A) is a representative flow cytometry dot-plotof drawn blood, stained for human glycophorin A (y-axis) and CFSE(x-axis), in which human cultured cells are double-positive. (FIG. 9B)is a plot of the clearance rate over time as a percentage ofdouble-positive cells remaining after NSG mice were injected with withhuman red blood cells (solid circles), cultured enucleated erythroidcells (dashed diamonds), cultured enucleated erythroid cells thatexpress an intracellular exogenous protein (dotted squares) and culturedenucleated erythroid cells that express a surface exogenous protein(open triangles).

FIG. 10A-FIG. 10D is an assessment of adverse events following injectionof cultured human erythroid cells into a mouse. (FIG. 10A-FIG. 10B) showlevels of (FIG. 10A) fibrinopeptide A and (FIG. 10B) fibrinopeptide Bassessed by ELISA in plasma collected from mice 20 minutes (black), 6hours (gray), and 48 hours (white) after injection with (1) human redblood cells, (2) cultured human erythroid cells, (3) cultured humanerythroid cells expressing an exogenous cytoplasmic protein, (4)cultured human erythroid cells expressing an exogenous surfacetransgene, or (5) recombinant protein. (FIG. 10C-FIG. 10D) showmicroscope images of histologically stained sections of spleen for miceinjected with (FIG. 10C) cultured human erythroid cells and (FIG. 10D)recombinant protein.

FIG. 11A-FIG. 11B tracks the expression of exogenous protein on culturederythroid cells in circulation. (FIG. 11A) is flow cytometry data ofblood drawn from a mouse that was injected with cultured human erythroidcells expressing an exogenous surface protein, showing the the percentof cultured human erythroid cells that are HA-positive over time. (FIG.11B) is a Western blot of blood drawn from two mice, wherein one mousewas injected with cultured human erythroid cells expressing an exogenouscytoplasmic protein, and wherein the other mouse was injected with thepurified recombinantly-produced exogenous protein in the absence of anycells, showing the level of HA-containing protein in the blood overtime.

FIG. 12A-FIG. 12C is an assessment of expansion and differentiation ofcultured human erythroid cells. (FIG. 12A) is a plot of expansion ratesfor distinct cultures of in vitro differentiated erythroid cells thatcontain transgenes (dashed line and dotted line) and cells that do notcontain a transgene (solid line). (FIG. 12B) is a flow cytometry plot ofcell surface markers GPA and CKIT for distinct cultures of culturedhuman erythroid cells that do not (left) or do (right) contain atransgene. (FIG. 12C) is a flow cytometry plot of cultured humanerythroid cells that do not (left) or do (right) contain a transgene,wherein the cells are stained with DNA stain DRAQ5 (y-axis) andanti-glycophorin A (x-axis), which identifies distinct populations of(1) enucleated cells, (2) nucleated cells, and (3) nuclei.

FIG. 13A is a schematic of a synthetic membrane-receiver complexcomprising a receiver polypeptide. The left panel depicts the flux of atarget substrate across the membrane of the synthetic membrane-receivercomplex. The target substrate is altered by an internally localizedenzymatic receiver polypeptide and the resulting product of theenzymatic reaction either remains in the synthetic membrane-receivercomplex or exits through the membrane. The right panel depicts asynthetic membrane-receiver complex that contains at least two receivers(e.g., receiver polypeptides), one being localized on the surface andone being internally localized. In this example, the surface-localizedreceiver aids a substrate to enter the synthetic membrane-receivercomplex, e.g., by carrying out a transporter function. The secondreceiver, localized internally, alters the substrate enzymatically. Theresulting product of the enzymatic reaction either remains in thesynthetic membrane-receiver complex or exits through the membrane,optionally aided by the first surface-localized receiver.

FIG. 13B is a schematic of another synthetic membrane-receiver complexcomprising a receiver polypeptide. FIG. 13B depicts a receiverpolypeptide localized on the surface of the synthetic membrane-receivercomplex. As shown, a target substrate can be acted upon directly by thereceiver. In the exemplified configuration, the target substrate doesnot need to cross the membrane to be enzymatically converted to aproduct. Optionally, the surface-localized enzymatic receiverpolypeptide can be made cleavable, e.g., if the complex enters aspecific microenvironment. In that instance, the receiver polypeptidewill be cleaved and become active in the extracellular space.

FIG. 13C is a schematic of yet another synthetic membrane-receivercomplex comprising a receiver. FIG. 13C depicts the lysis of a syntheticmembrane-receiver complex containing internally-localized receiver(e.g., a polypeptide) and optional payload (e.g., a therapeutic agent)which may result from a variety of stimuli. Upon lysis, theinternally-localized receiver and optional payload is released into themicroenvironment where it may act on a target substrate.

FIG. 14A is a schematic of three ways in which a receiver may belocalized in a synthetic membrane-receiver complex. FIG. 14B is aschematic of three ways in which a receiver localized in or on asynthetic membrane-receiver complex may act on a target in circulation.FIG. 14C is a schematic of an auto-catalytic fusion of an endogenouspolypeptide anchor to a receiver utilizing a SpyTag-SpyCatchermechanism.

DETAILED DESCRIPTION OF THE INVENTION

Therapeutic technologies attempting to employ circulating agents havebeen developed in the past to address some of the challenges indelivering treatments such as pharmaceutical drugs to patients. Nonepossess one or many of the features and benefits of the syntheticmembrane-receiver complexes provided herein. Aspects of the inventionprovide compositions capable of multiple, distinct utilities, whichutilize biochemical and biophysical mechanisms not previously addressed.Aspects of the invention relate to compositions and methods forperforming, e.g., functions related to circulating clearance andfunctions related to metabolic enzyme delivery, and methods for treatingor preventing a variety of diseases, disorders and conditions.Accordingly, the compositions and methods disclosed herein address thelong sought after need for therapeutic compositions that are distributedthrough the circulatory system that have increased half-life, safetyprofile, and/or efficacy that avoid shortcomings associated withprevious approaches such as undesirable immunological reactions, shorthalf-life due to rapid clearance from the circulation, and off-targeteffects, among others.

Functions related to circulating clearance include activitiescharacterized by, e.g., the specific binding, degradation, and/orsequestration of a target (e.g., a pathogenic substance or toxicmolecule) in the circulatory system of a subject by a syntheticmembrane-receiver complex comprising a receiver capable of interactingwith a target as described herein. Synthetic membrane-receiver complexesare introduced or capable of being introduced into the circulation of asubject. In some embodiments, the bound or sequestered targets areguided to the liver, spleen, or any other site in which they may beremoved from the circulatory system.

Functions related to metabolic enzyme delivery include activitiescharacterized by, e.g., removal of a target (e.g., a pathogenicsubstance or toxic molecule), in circulation of a subject by a syntheticmembrane-receiver complex as described herein that comprises, e.g., oneor more metabolic enzyme receiver polypeptides within the complex or onthe surface of the complex, such that the receiver polypeptide interactswith and modifies the target. Modification of the target includes, e.g.,alteration of the bioavailability of the target, cleaving, degrading,and/or otherwise inactivating the target by the receiver. In someembodiments, the enzymatic polypeptide is protected from the immunesystem. In some embodiments, the half-life of the enzyme is extendedand/or an immunogenic reaction is reduced when administered in thesubject.

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Many modifications and other embodiments of the inventions set forthherein will easily come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural references unless the contentclearly dictates otherwise.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, any concentration range, percentage range, ratio range,or integer range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated.

“Comprise,” “comprising,” and “comprises” and “comprised of” as usedherein are synonymous with “include”, “including”, “includes” or“contain”, “containing”, “contains” and are inclusive or open-endedterms that specifies the presence of what follows e.g. component and donot exclude or preclude the presence of additional, non-recitedcomponents, features, element, members, steps, known in the art ordisclosed therein.

As used herein, the terms “such as”, “for example” and the like areintended to refer to exemplary embodiments and not to limit the scope ofthe present disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, preferred materialsand methods are described herein.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventorsdescribed herein are not entitled to antedate such disclosure by virtueof prior invention or for any other reason.

Definitions

“Administration,” “administering” and variants thereof means introducinga composition, such as a synthetic membrane-receiver complex, or agentinto a subject and includes concurrent and sequential introduction of acomposition or agent. The introduction of a composition or agent into asubject is by any suitable route, including orally, pulmonarily,intranasally, parenterally (intravenously, intramuscularly,intraperitoneally, or subcutaneously), rectally, intralymphatically, ortopically. Administration includes self-administration and theadministration by another. A suitable route of administration allows thecomposition or the agent to perform its intended function. For example,if a suitable route is intravenous, the composition is administered byintroducing the composition or agent into a vein of the subject.Administration can be carried out by any suitable route,

“Anchor” or “anchor domain” or “A domain” is used to refer to theportion of a receiver polypeptide, including a fusion or chimericreceiver polypeptide that is in contact with the lipid layer of asynthetic membrane-receiver polypeptide complex. The receiverpolypeptide may interact with the lipid layer via a phospholipid tailinsertion, covalent binding to a lipid layer constituent, an ionic bond,hydrogen bond, or via a single or multi-pass transmembrane polypeptidedomain that cross one or more of the lipid layers.

As used herein, the term “antibody” encompasses an immunoglobulinwhether natural or partly or wholly synthetically produced, andfragments thereof. The term also covers any protein having a bindingdomain which is homologous to an immunoglobulin binding domain. Theseproteins can be derived from natural sources, or partly or whollysynthetically produced. “Antibody” further includes a polypeptidecomprising a framework region from an immunoglobulin gene or fragmentsthereof that specifically binds and recognizes an antigen. Use of theterm antibody is meant to include whole antibodies, polyclonal,monoclonal and recombinant antibodies, fragments thereof, and furtherincludes single-chain antibodies, humanized antibodies; murineantibodies; chimeric, mouse-human, mouse-primate, primate-humanmonoclonal antibodies, anti-idiotype antibodies, antibody fragments,such as, e.g., scFv, (scFv)2, Fab, Fab′, and F(ab′)2, F(ab 1)2, Fv, dAb,and Fd fragments, diabodies, and antibody-related polypeptides. Antibodyincludes bispecific antibodies and multispecific antibodies so long asthey exhibit the desired biological activity or function.

The term “antigen binding fragment” used herein refers to fragments ofan intact immunoglobulin, and any part of a polypeptide includingantigen binding regions having the ability to specifically bind to theantigen. For example, the antigen binding fragment may be a F(ab′)2fragment, a Fab′ fragment, a Fab fragment, a Fv fragment, or a scFvfragment, but is not limited thereto. A Fab fragment has one antigenbinding site and contains the variable regions of a light chain and aheavy chain, the constant region of the light chain, and the firstconstant region CH1 of the heavy chain. A Fab′ fragment differs from aFab fragment in that the Fab′ fragment additionally includes the hingeregion of the heavy chain, including at least one cysteine residue atthe C-terminal of the heavy chain CH1 region. The F(ab′)2 fragment isproduced whereby cysteine residues of the Fab′ fragment are joined by adisulfide bond at the hinge region. A Fv fragment is the minimalantibody fragment having only heavy chain variable regions and lightchain variable regions, and a recombinant technique for producing the Fvfragment is well known in the art. Two-chain Fv fragments may have astructure in which heavy chain variable regions are linked to lightchain variable regions by a non-covalent bond. Single-chain Fv (scFv)fragments generally may have a dimer structure as in the two-chain Fvfragments in which heavy chain variable regions are covalently bound tolight chain variable regions via a peptide linker or heavy and lightchain variable regions are directly linked to each other at theC-terminal thereof. The antigen binding fragment may be obtained using aprotease (for example, a whole antibody is digested with papain toobtain Fab fragments, and is digested with pepsin to obtain F(ab′)2fragments), and may be prepared by a genetic recombinant technique. AdAb fragment consists of a VH domain. Single-chain antibody moleculesmay comprise a polymer with a number of individual molecules, forexample, dimmer, trimer or other polymers.

“Applicator” refers to any device used to connect to a subject. Thisincludes, e.g., needles, cannulae, catheters, and tubing.

“Associated with” when used to describe the relationships among multiplecompounds or molecules encompasses such as, e.g., any interactionbetween a receiver and a target or between a synthetic membrane-receivercomplex and a target. This includes enzymatic interaction, ionicbinding, covalent binding, non-covalent binding, hydrogen bonding,London forces, van der Waals forces, hydrophobic interaction, lipophilicinteractions, magnetic interactions, electrostatic interactions, and thelike.

“Associated with” when used to describe the relationships among atarget, entity, compound, agent, or molecule and a disease, disorder,condition, symptom or phenotype is any link that may reasonably be madebetween them, including a causal link, or a statistical significantlink, an empirically established link, a suggested link, whether or notcausative of the disease, disorder, condition, symptom or phenotype.

“Autoimmune disorders” generally are conditions in which a subject'simmune system attacks the body's own cells, causing tissue destruction.Autoimmune disorders may be diagnosed using blood tests, cerebrospinalfluid analysis, electromyogram (measures muscle function), and magneticresonance imaging of the brain, but antibody testing in the blood, forself-antibodies (or auto-antibodies) is particularly useful. Usually,IgG class antibodies are associated with autoimmune diseases.

“Binding” describes an interaction among compounds or molecules, e.g.,between a receiver and a target or between a synthetic membrane-receivercomplex and a target, that comes about by covalent binding ornon-covalent binding, including ionic binding, electrostaticinteractions, hydrogen bonding, London forces, van der Waals forces,hydrophobic interaction, lipophilic interactions, and similar.

The “biological activity of a polypeptide” refers to any molecularactivity or phenotype (such as, e.g., binding, signal transduction,catalytic, etc.) that is caused by the polypeptide, such as a receiverpolypeptide.

As used herein, the term “biological sample” refers to any type ofmaterial of biological origin isolated from a subject, including, forexample, DNA, RNA, lipids, carbohydrates, and protein. The term“biological sample” includes tissues, cells and biological fluidsisolated from a subject. Biological samples include, e.g., but are notlimited to, whole blood, plasma, serum, semen, saliva, tears, urine,fecal material, sweat, buccal, skin, cerebrospinal fluid, bone marrow,bile, hair, muscle biopsy, organ tissue or other material of biologicalorigin known by those of ordinary skill in the art. Biological samplescan be obtained from, e.g., biopsies of internal organs or from cancers.Biological samples can be obtained from subjects for diagnosis orresearch or can be obtained from healthy subjects, as controls or forbasic research.

The “clearance rate” as used herein is calculated by measuring theamount or concentration of, e.g., target, receiver, target-receiver, orsynthetic membrane-receiver complexes remaining in the circulatorysystem of a subject over time. For example, 1%, 2%, 3%, 4%, 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of targetdetected in a first sample may still be detected in a second sample thatis taken 1 hour, 5 hours, 10 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 2 years, 3 years, 4 years, or 5 years later. Theclearance rate may alternatively be expressed as: number of entities(e.g., target/receiver) per unit of time (e.g., per day). An increase inclearance rate is a rate greater than that exhibited in an untreated orhealthy suitable control subject. A decrease in clearance rate is a rateless than that exhibited in an untreated or healthy suitable controlsubject. The increase or decrease may be 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, 1000% ormay be 1.1-fold, 1.2-fold, 1.3 fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or1000-fold. An increase in clearance rate of a target includes, e.g., aslow down in the accumulation of a target, a reaching of a newequilibrium of generation and degradation, and a reversal of anaccumulation, e.g., a decrease in the number or concentration of thetarget in circulation.

“Cleaving” as used herein is a process that disrupts a bondinginteraction present in a target, such as a polypeptide or nucleic e.g.,to produce two or more entities that after cleaving can be separatedfrom one another. The separation can involve, e.g., disrupt an ionicbond, a covalent bond, a polar covalent bond, a non-polar covalent bond,or a metallic bond. As cleaving applies to polypeptide targets, cleavagecan involve breaking one or more peptide bonds. As cleaving applies tosmall molecule targets, cleavage can involve breaking one or more carbonor sulfide bonds. As cleaving applies to nucleotide sequences, cleavagecan involve breaking one or more phosphodiester bonds. As cleavingapplies to microbes such as bacteria, fungi, or viruses, cleavage caninvolve lysis of a membrane or capsid structure. Cleaving can be carriedout by an enzyme, e.g., a catalytically active receiver polypeptide.Receivers can comprise, e.g., exonuclease, endonuclease, or proteaseactivity.

The “circulatory system of a subject,” as used herein, encompasses thespace occupied by whole blood and optionally the lymphatic system in ahuman, inclusive of plasma and all circulating cells and molecules, anddistributed throughout arteries, veins, capillaries, and lymphaticvessels of all tissues. The “circulatory concentration” is theconcentration of a target, e.g., a cell, polypeptide (such as anantibody, pathogenic antigen, etc.), therapeutic agent, small molecule,metabolite or other entity, a receiver or a synthetic membrane-receivercomplex in the space defined as the circulatory system. In certainembodiments, the concentration may be defined as the number of free(unbound) entities in a given volume. In other embodiments, theconcentration may be defined as the total number of entities in a givenvolume.

The term “complementarity determining region (CDR)” used herein refersto an amino acid sequence found in the variable region of a heavy chainor a light chain of an immunoglobulin. The CDRs determine thespecificity of an antibody and may provide a contact residue for bindingto a specific epitope of an antigen. The heavy chain and the light chainmay respectively include three CDRs (CDRH1, CDRH2, and CDRH3, and CDRL1,CDRL2, and CDRL3). Four framework regions, which have more highlyconserved amino acid sequences than the CDRs, separate the CDR regionsin the VH or VL.

A “complex” as used herein comprises an association of two or moreentities. A complex may comprise one or more polypeptides, nucleic acid,lipids, carbohydrates, inorganic compounds, organic compounds, and thelike. A complex can be functional (multiunit polypeptides) ornon-functional (e.g., aggregates or precipitates) and may havebeneficial or detrimental properties (e.g., immune complexes). Complexesmay be naturally occurring or may be man-made or synthetic. Syntheticcomplexes include higher order entities, e.g., subcellular structuresand cells if they comprise a synthetic compound or molecule. Forexample, a synthetic membrane-receiver complex includes a cellcomprising a receiver.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.As used herein the term “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine.

“Decrease,” in the context of a symptom of a treated disease, disorderor condition, refers to a reduction in measurable or conveyableparameters associated with the disease or condition that manifest assymptoms. Examples of measurable parameters are a reduction in thesubject's body temperature, a reduction in the concentration of targetsin a sample taken from the subject, reduction in the intensity ofinflammation or size of an inflamed area, reduction in the number ofinfiltrating cells, reduction in the number of episodes associated withthe disease, disorder or condition, increase/decrease in organ size,weight gain/loss, etc. Examples of conveyable parameters are, e.g., thesubject's own assessment of well being and quality of life. For example,for self-antibody mediated diseases, the decrease may be quantified asone, or a combination of, the following parameters: reducedinflammation, reduced flare-ups, reduced fatigue, reduced bloodclotting, reduced swelling, increased energy, or increased hair growth,etc. The parameters that may be quantified are those appropriate forassessing the specific disease, disorder or condition that is beingtreated. Delay, in the context of symptoms of a treated disease,disorder or condition, refers to the significant extension of amanageable health condition that would otherwise become exacerbated,using a treatment.

“Degrading” is defined as the process in which a target is eitherdirectly, or indirectly, reduced, inactivated, decomposed,deconstructed, lysed, dissolved, broken, lessened, impaired, weakened,deteriorated, diminished, or partitioned.

“Different polypeptide origin” refers to the organism or species fromwhich a genetic sequence encoding the polypeptide, the polypeptide, orportion thereof, is sourced. In certain embodiments, a fusion comprisingpolypeptides of different polypeptide origin may include a receiverpolypeptide that is encoded by the genetic sequence for human adenosinedeaminase and the genetic sequence for phenylalanine hydroxylase fromchromobacterium violaceum.

A “domain” is a part of a polypeptide, such as a receiver polypeptidethat is generally having a 3-dimensional structure and may exhibit adistinct activity, function, such as, e.g., a catalytic, an enzymatic, astructural role, or a binding function.

Duration refers to the period of time that a portion of the syntheticmembrane-receiver polypeptide complex exists in a specific tissue or anorganism as a whole. This applies to 0.1% 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% of the initial dose or concentration ofthe synthetic membrane-receiver polypeptide complex. In someembodiments, the synthetic membrane-receiver complex is formulated forlong-term duration. In some embodiments, the synthetic membrane-receivercomplex is formulated for short-term duration.

By an “enriched population of cells” it is meant a population of cellsthat is substantially comprised of a particular cell of interest. In anenriched population, 50% or more of the cells in the population are thecells of interest, e.g., 50%, 60%, 70%, usually 80%, 85%, 90%, moreusually 92%, 95%, 96%, 97%, 98%, or 99%, sometimes as much as 100% ofthe cells in the population. The separation of cells of interest from acomplex mixture or heterogeneous culture of cells may be performed byany convenient means known in the art, for example, by affinityseparation techniques such as magnetic separation using magnetic beadscoated with an affinity reagent, affinity chromatography, or “panning”with an affinity reagent attached to a solid matrix, e.g., plate, orother convenient technique. Other techniques providing accurateseparation include fluorescence activated cell sorters, which can havevarying degrees of sophistication, such as multiple color channels, lowangle and obtuse light scattering detecting channels, impedancechannels, etc. The cells may be selected against dead cells by employingdyes associated with dead cells. Any technique may be employed which isnot unduly detrimental to the viability of the desired cells.

“Enucleation” is the rendering of a cell to a non-replicative state,either through inactivation or removal of the nucleus.

An “epitope” includes any segment on an antigen to which an antibody orother ligand or binding molecule binds. An epitope may consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. In someembodiments, receivers comprise specific epitopes. In some embodiments,targets comprise specific epitopes.

“Erythroid cells” as used herein, include nucleated red blood cells, redblood cell precursors, and enucleated red blood cells and those listedin Table 2. For example, the erythroid cells are a cord blood stem cell,a CD34+ cell, a hematopoietic stem cell (HSC), a spleen colony forming(CFU-S) cell, a common myeloid progenitor (CMP) cell, a blastocytecolony-forming cell, a burst forming unit-erythroid (BFU-E), amegakaryocyte-erythroid progenitor (MEP) cell, an erythroidcolony-forming unit (CFU-E), a reticulocyte, an erythrocyte, an inducedpluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), apolychromatic normoblast, an orthochromatic normoblast, or a combinationthereof. In some embodiments, the erythroid cells are immortal orimmortalized cells. For example, immortalized erythroblast cells can begenerated by retroviral transduction of CD34+ hematopoietic progenitorcells to express Oct4, Sox2, Klf4, cMyc, and suppress TP53 (e.g., asdescribed in Huang et al., Mol Ther 2013, epub ahead of print September3). In addition, the cells may be intended for autologous use or providea source for allogeneic transfusion. Erythroid cells can be contactedwith a receiver to generate a synthetic membrane-receiver complex.Erythroid cells comprising a receiver are one example of a syntheticmembrane-receiver complex. In some embodiments, erythroid cells arecultured. In some embodiments, erythroid progenitor cells are contactedwith a receiver to generate a synthetic membrane-receiver complex.

As used herein, the term “excipient” refers to an inert substance addedto a pharmaceutical composition to further facilitate administration ofa compound. Examples of excipients include, but are not limited to,calcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, anti-coagulants,and polyethylene glycols.

The receiver, including a receiver polypeptide is “exogenous” or“heterologous”, thus it may either not naturally exist, such as a fusionor chimera comprising domains of different polypeptide or speciesorigin, it may not naturally occur in a naturally occurring cell, suchas an unmodified erythrocyte or platelet, it may not function in thesame way as a naturally occurring polypeptide would, or it may notnaturally occur in the quantity that the receiver polypeptide occurs,e.g., in embodiments in which the synthetic membrane-receiverpolypeptide complex is a cell-derived polypeptide receiver that isoverexpressed as compared to the expression of a naturally occurringpolypeptide in an unmodified cell. In some embodiments, the polypeptidereceiver is expressed from an exogenous nucleic acid. In someembodiments, the receiver is isolated from a source and loaded into orconjugated to a synthetic membrane-receiver complex.

The term “exogenous” when used in the context of nucleic acid includes atransgene and recombinant nucleic acids.

As used herein, the term “expression” refers to the process to produce apolypeptide, such as a receiver polypeptide including transcription andtranslation. Expression may be, e.g., increased by a number ofapproaches, including: increasing the number of genes encoding thepolypeptide, increasing the transcription of the gene (such as byplacing the gene under the control of a constitutive promoter),increasing the translation of the gene, knocking out of a competitivegene, or a combination of these and/or other approaches.

A synthetic membrane-receiver complex that is “formulated for long-termduration” is, in some embodiments, one that is part of a population ofsynthetic membrane-receiver complexes wherein a substantial fraction ofthe population resides in the circulatory system for more than 10 days,e.g., 15, 21, 25, 35, 45, 50, 60, 90, 100, 110, or 120 days. In someembodiments, the population may have an increased half-life, e.g., 1.5×,2×, 5×, 10×, 20×, 50×, 100× more time in circulation, when formulatedfor long-term duration compared to the duration exhibited by apopulation of unformulated complexes. In some embodiments, an entitysuch as a receiver may have an increased half-life, e.g., 1.5×, 2×, 5×,10×, 20×, 50×, 100× more time in circulation, when formulated forlong-term duration compared to the duration that entity would exhibit inan unmodified state.

A synthetic membrane-receiver complex that is “formulated for short-termduration” is, in some embodiments, one that is part of a population ofsynthetic membrane-receiver complexes wherein a substantial fraction ofthe population resides in the circulatory system for less than 10 days,e.g., 9, 8, 7, 6, 5, 4, 3, 2 days, 1 day, 12 hours, or 6 hours. In someembodiments, the population may have a decreased half-life, e.g., 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% less time incirculation, when formulated for short-term duration compared to theduration exhibited by a population of unformulated complexes. In someembodiments, an entity such as a receiver may have a reduced half-life,e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% less time incirculation, when formulated for short-term duration compared to theduration that entity would exhibit in an unmodified state.

“Formulated for residency in the circulatory system”, as used herein,describes one or more modifications to an entity, such as a syntheticmembrane-receiver complex formulated for administration to thecirculatory system of a subject that substantially decrease recognition,modification, degradation, and/or destruction of the entity bycomponents of the circulatory system (e.g., circulating immune cells,antibodies, enzymatic activities) thereby increasing the half-life ofthe entity when compared to an unmodified entity.

A “functional” receiver or synthetic membrane-receiver complex refers toa synthetic membrane-receiver complex or a receiver that exhibits adesired or specified activity or characteristic, including enzymatic,catalytic or metabolic activity, structural integrity, immunogeniccomplementarity, target binding, and correct localization or is capableof promoting a desired or specified effect or phenotype.

“Fusion or chimera” is defined as a polypeptide sequence, orcorresponding encoding nucleotide sequence, that is derived from thecombination of two or more sequences that are not found together innature. This may be a combination of separate sequences derived fromseparate genes within the same genome, or from heterologous genesderived from distinctly different species' genomes.

“Genetic material” refers to nucleic acid molecules having nucleotidesequences of adenosine, thymine, uracil, cytosine, and guanine capableof encoding a gene.

The term “heavy chain” used herein is understood to include afull-length heavy chain including a variable region (VH) having aminoacid sequences that determine specificity for antigens and a constantregion having three constant domains (CH1, CH2, and CH3), and fragmentsthereof. In addition, the term “light chain” used herein is understoodto include a full-length light chain including a variable region (VL)having amino acid sequences that determine specificity for antigens anda constant region (CL), and fragments thereof.

The term “homolog” indicates polypeptides, including receiverpolypeptide that have the same or conserved residues at a correspondingposition in their primary, secondary or tertiary structure. Functionalhomologs include receivers and other polypeptides that exhibit similarfunction and/or specificity (e.g., for a particular target).

A naturally occurring intact antibody, or immunoglobulin, includes fourpolypeptides: two full-length light chains and two full-length heavychains, in which each light chain is linked to a heavy chain bydisulfide bonds. Each heavy chain has a constant region and a variableregion. Similarly, each light chain has a constant region and a variableregion. There are five heavy chain classes (isotypes): gamma (γ), mu(μ), alpha (α), delta (δ), or epsilon (ε), and additionally severalsubclasses gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha1(α1), and alpha 2(α2). The light chain constant region can be eitherkappa (κ) or lambda (λ) type. The variable regions differ in sequenceamong antibodies and are used in the binding and specificity of a givenantibody to its particular antigen.

As used herein, the term “increase,” “enhance,” “stimulate,” and/or“induce” (and like terms) generally refers to the act of improving orincreasing, either directly or indirectly, a concentration, level,function, activity, or behavior relative to the natural, expected, oraverage, or relative to a control condition.

As used herein, the term “inhibit,” “suppress,” “decrease,” “interfere,”and/or “reduce” (and like terms) generally refers to the act ofreducing, either directly or indirectly, a concentration, level,function, activity, or behavior relative to the natural, expected, oraverage, or relative to a control condition.

A “library” as used herein includes a collection of nucleic acidmolecules (e.g., DNA, RNA) having diverse nucleic acid sequences, agenetically diverse collection of clones, a collection of diversepolypeptides, a diverse collection of cells, etc.

As used herein, “a mammalian subject” includes all mammals, includingwithout limitation, humans, domestic animals (e.g., dogs, cats and thelike), farm animals (e.g., cows, sheep, pigs, horses and the like) andlaboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs andthe like). The terms “individual,” “subject,” “host,” and “patient,” areused interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans. Themethods described herein are applicable to both human therapy andveterinary applications. In some embodiments, the subject is a mammal,and in other embodiments the subject is a human.

“Medical device” refers to any device, apparatus or machine used todeliver a dose of a synthetic membrane-receiver complex and/or atherapeutic agent. This includes containers, bottles, vials, syringes,bags, cartridges, cassettes, magazines, cylinders, or canisters.

“Medical kit” refers to a packaged unit that includes a medical device,applicator, appropriate dosage of synthetic membrane-receiver complexoptionally including a therapeutic agent, and relevant labeling andinstructions.

As used herein, the term “modulate,” “modulating”, “modify,” and/or“modulator” generally refers to the ability to alter, by increase ordecrease, e.g., directly or indirectlypromoting/stimulating/upregulating or interferingwith/inhibiting/downregulating a specific concentration, level,expression, function or behavior, such as, e.g., to act as an antagonistor agonist. In some instances a modulator may increase and/or decrease acertain concentration, level, activity or function relative to acontrol, or relative to the average level of activity that wouldgenerally be expected or relative to a control level of activity.

“Membrane” as used herein is a boundary layer that separates an interiorspace from an exterior space comprising one or more biologicalcompounds, typically lipids, and optionally polypeptides. Membranes canbe lipid bilayers. In certain embodiments, membranes comprise one ormore of phosphatidylcholine, sphingomyelin, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, orphosphatidic acid. In some embodiments, membranes comprise one or morepolypeptides such as ankyrin and coenzyme Q10. Included in thedefinition of membrane are cell membranes comprising, e.g., aphospholipid bilayer and cell membrane associated polypeptides. Thesynthetic membrane-receiver complex comprises a membrane as definedherein.

The phrase “nucleic acid molecule” refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases. It includeschromosomal DNA and self-replicating plasmids, vectors, mRNA, tRNA,siRNA, etc. which may be recombinant and from which exogenouspolypeptides may be expressed when the nucleic acid is introduced into acell.

Orthologs are defined as genes in different species that evolved from acommon ancestral gene by speciation.

The term “pharmaceutically-acceptable” and grammatical variationsthereof, refers to compositions, carriers, diluents and reagents capableof administration to or upon a subject without the production ofundesirable physiological effects to a degree that would prohibitadministration of the composition. For example,“pharmaceutically-acceptable excipient” includes an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans, as well as any carrier or diluent that does not causesignificant irritation to a subject and does not abrogate the biologicalactivity and properties of the administered compound.

Some agents may be administered as “pharmaceutically acceptable salt”,e.g., prepared from inorganic and organic acids. Salts derived frominorganic acids include hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. Salts derived fromorganic acids include acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Salts can also beprepared from inorganic and organic bases. Salts derived from inorganicbases, include by way of example only, sodium, potassium, lithium,ammonium, calcium and magnesium salts. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary andtertiary amines Any ordinary skilled person in the art will know how toselect a proper pharmaceutically acceptable carrier, a pharmaceuticallyacceptable salt thereof for implementing this invention without undueexperimentation.

As used herein, the term “pharmaceutical composition” refers to one ormore of the compounds described herein, such as, e.g., a syntheticmembrane-receiver polypeptide complex mixed or intermingled with, orsuspended in one or more other chemical components, such asphysiologically acceptable carriers and excipients. One purpose of apharmaceutical composition is to facilitate administration of a compoundto a subject.

Certain embodiments provide various polypeptide molecules havingsequences associated with a desired function or activity, such asreceiver polypeptides. A polypeptide is a term that refers to a chain ofamino acid residues, regardless of post-translational modification(e.g., phosphorylation or glycosylation) and/or complexation withadditional polypeptides, synthesis into multisubunit complexes, withnucleic acids and/or carbohydrates, or other molecules. Proteoglycanstherefore also are referred to herein as polypeptides. In certainembodiments, the synthetic membrane-receiver complex comprises apolypeptide receiver and is referred to a “synthetic membrane-receiverpolypeptide complex.” In certain embodiments, the syntheticmembrane-receiver complex comprises one or more non-receiverpolypeptides that are optionally membrane-associated and that exhibitcatalytic and/or metabolic activity independent of the receiver. Forexample, the non-receiver polypeptides may have catalytic activity foran organic compound including a metabolite. In certain embodiments, thesynthetic membrane-receiver complex comprises a sufficient number ofnon-receiver polypeptides (and optionally non-protein co-factors) tosupport a metabolic pathway.

The term “pharmaceutically active agent” or “pharmaceutical agent” isdefined as any compound, e.g., a small molecule drug, or a biologic(e.g., a polypeptide drug or a nucleic acid drug) that when administeredto a subject has a measurable or conveyable effect on the subject, e.g.,it alleviates or decreases a symptom of a disease, disorder orcondition. In some embodiments, the pharmaceutical agent may beadministered prior to, in combination with, or following the delivery ofa synthetic membrane-receiver polypeptide complex. In some embodiments,the pharmaceutically active agent exerts a synergistic treatment effectwith the synthetic membrane-receiver polypeptide complex. In someembodiments, the pharmaceutically active agents exerts an additivetreatment effect with the synthetic membrane-receiver polypeptidecomplex.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of an operably linked nucleic acid. Promotersinclude necessary nucleic acid sequences near the start site oftranscription. A promoter also optionally includes distal enhancer orrepressor elements. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

A “receiver,” as used herein, is an entity capable of interacting with atarget, e.g., to associate with or bind to a target. A receiver cancomprise or can consist essentially of a polypeptide. In someembodiments, the receiver comprises a polypeptide, a carbohydrate, anucleic acid, a lipid, a small molecule, or a combination thereof. Inembodiments in which a receiver is a naturally occurring compound ormolecule, the receiver is “synthetic” in the sense that it is anexogenous or heterologous compound or molecule with regard to itspresence in the synthetic membrane-receiver complex. In otherembodiments the receiver is “synthetic” in the sense that it is aman-made compound or molecule, such as a fusion or chimera, anon-naturally occurring polypeptide, carbohydrate, nucleic acid, lipid,or combination thereof, or a man-made small molecule or othertherapeutic agent. For example, the receiver may comprise a fusion orchimera comprising one or more of an S domain, an A domain and a Udomain. The S domain is a surface domain exposed to the environmentaround the synthetic membrane-receiver complex, such as the circulatorysystem of a subject. The A domain is an anchor domain that attaches theS domain to the synthetic membrane of the synthetic membrane-receiverpolypeptide complex. The U domain faces the unexposed side of or islocated within the synthetic membrane-receiver complex, i.e. the sidethat is not exposed to the external environment of the circulatorysystem of a subject. Irrespective of any domains, a receiver may belocated on the surface of the synthetic membrane-receiver polypeptidecomplex or may be located within the complex. The receiver may beassociated with the membrane of the synthetic membrane-receiver complex,e.g., the receiver is anchored in, conjugated to or otherwise bound tothe membrane. In some embodiments, the receiver may be conjugated to themembrane of the synthetic membrane-receiver complex by chemical orenzymatic conjugation. In other embodiments, the receiver is notconjugated to the membrane. In some embodiments, the receiver is notassociated with the membrane of the synthetic membrane-receiver complexand is located within the membrane-encapsulated volume of the complex.In some embodiments, a receiver located within the syntheticmembrane-receiver complex does not substantially diffuse out of thecomplex and/or may not permeate the membrane. In other embodiments, thereceiver may substantially diffuse out of the complex and/or maypermeate the membrane. In some embodiments, the receiver is loaded,e.g., introduced into or put onto the synthetic membrane-receivercomplex. A receiver that is loaded is not biologically synthesized bythe synthetic membrane-receiver complex. A receiver suitable for loadingmay be e.g., produced in a cell-based expression system, isolated from abiological sample, or chemically or enzymatically synthesized, and thenloaded into or onto the synthetic membrane-receiver complex. In someembodiments, the receiver may be further modified by the syntheticmembrane-receiver complex after loading. In other embodiments, thereceiver is not modified after loading. In some embodiments, thereceiver polypeptide is not loaded onto or into the complex. In someembodiments, the receiver is made, e.g., biologically synthesized by thesynthetic membrane-receiver complex. Typically a receiver polypeptide isexpressed by the synthetic membrane-receiver complex from an exogenousnucleic acid molecule (e.g., a DNA or mRNA) that was introduced into thecomplex. The receiver may bind to and/or sequester a target.Alternatively or in addition the receiver may exhibit a catalyticactivity toward the target, e.g., the receiver may convert or modify thetarget, or may degrade the target. A product may then optionally bereleased from the receiver.

“Residency” of a synthetic membrane-receiver complex refers to theperiod of time it spends in a physiological location. The specificlocation of the synthetic membrane-receiver complex may change duringits lifetime and “residency” applies to the period of time spent invarious environments, including vascular circulation, peripheraltissues, capillaries, digestive system, pulmonary system, nasal tissues,epidermal surface, and interstitial tissue. In specific embodiments, thesynthetic membrane-receiver complex resides in the circulatory system ofa subject.

“Replicating nucleic acid” refers to deoxyribonucleic acid (DNA) that iscapable of being copied by enzymes dedicated to the increasing thenumber of copies of the DNA. Usually, DNA replication leads to theproduction of two identical replicas from one original DNA molecule. DNAreplication comprises the incorporation of nucleotides into a growingDNA strand by DNA polymerase matched to the template strand one at atime via the creation of phosphodiester bonds.

“Sequestering” is defined as cloistering, occluding, separating,segregating, hiding, insulating, or isolating of a target and preventingit from freely interacting with its environment.

“Specifically binding” or “specifically interacting”, as used herein,describes any interaction between two entities (e.g., a target with areceiver, such as an antibody with an antigen, a receptor with a ligand,an enzyme with a substrate, biotin with avidin, etc.) that is saturable,often reversible and so competitive, as these terms are understood bythose of ordinary skill in the chemical and biochemical arts. e.g.,Specific binding involving biological molecules such as, e.g., proteins,peptides and nucleic acid occurs when one member of the binding pair hasa site with a shape and distribution of charged, polar, or hydrophobicmoieties such that the interaction of the cognate ligand with that siteis characterized by favorable energetics (i.e., a negative free energyof binding). The specificity of the interaction may be measured orexpressed as a binding constant (Kd). The Kd may range from a mM rangeto a pM range, including μM ranges and nM ranges. Typical Kd values arebelow about 10⁻⁶M, below about 10⁻⁷ M, below about 10⁻⁸ M, and in someembodiments below about 10⁻⁹ M.

As used herein, the term “substantially” or “substantial” refers, e.g.,to the presence, level, or concentration of an entity in a particularspace, the effect of one entity on another entity, or the effect of atreatment. For example, an activity, level or concentration of an entityis substantially increased if the increase is 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold relative to a baseline.An activity, level or concentration of an entity is also substantiallyincreased if the increase is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, or 500% relative to a baseline. An entity may besubstantially present in a particular space if it can be detected bymethods known in the art. An entity may not be substantially present ina particular space if it is present at levels below the limit ofdetection for assays and methods known in the art. In some embodiments,an entity may not be substantially present in a particular space if itis barely detectable but only in non-functional quantities or minutequantities that do not cause or change a phenotype. In otherembodiments, an entity may not be substantially present in a particularpopulation if it is present and can be detected only in a small numberof constituents making up the population, e.g., less than 10%, 9%, 8%,7%, 6%, 5%, 4%, 3% 2% or less than 1%, 0.5%, 0.1% of constituents of thepopulation. For example, an exogenous nucleic acid may not be retainedupon enucleation, the cell is rendered non-replicative, and theenucleated cell is incapable of continued expression of the receiverpolypeptide encoded by the exogenous nucleic acid. The loss of theability of the cell to continue to significantly translate the exogenouspolypeptide “effectively terminates” protein expression. In certainembodiments, the synthetic membrane-receiver complex is substantiallyincapable of self-replication, e.g., the replication of nucleic acids.For example, the synthetic membrane-receiver polypeptide complex doesnot substantially incorporate a nucleoside if contacted with labelednucleoside, such as thymidine, in an incorporation assay. In someembodiments, the synthetic membrane-receiver polypeptide complex doesnot contain a substantial amount of self-replicating nucleic acids. Theterm “substantial identity” of polynucleotide or nucleic acid sequencesmeans that a polynucleotide comprises a sequence that has at least 25%sequence identity. Alternatively, percent identity can be any integerfrom 25% to 100%. More preferred embodiments include at least: 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%compared to a reference sequence using the programs described herein;preferably BLAST using standard parameters.

“Synthetic” refers to a compound or molecule that is either man-made andnon-naturally occurring, or if it is naturally occurring is placed in acontext or location that it would not naturally exist, or if itnaturally exists in the context or location is in a state of purity, oris present in an amount, concentration or number that it would notnaturally be present in the context or location. Synthetic entities canbe isolated or purified compounds that are optionally chemically orenzymatically modified from their natural state, exogenous nucleicacids, exogenous (heterologous) receivers, and the like. The presence ofa synthetic compound or molecule, as defined herein, in any entityrenders the entire entity “synthetic”. For example, a cell comprising areceiver is a synthetic cell.

A “target,” as used herein, is an entity capable of interacting with areceiver, e.g., to associate with or bind to a receiver. A “target”includes, but is not limited to a polypeptide (e.g., an antibody orantibody-related polypeptide, a complement constituent, an amyloidprotein, a pathogen, a toxin, a prion), a molecule (e.g., a metabolite,a steroid, a hormone, a carbohydrate; an oligosaccharide; a chemical; apolysaccharide, a DNA; an RNA; a lipid, an amino acid, an element, atoxin or pathogen), a complex (e.g., an immune complex), or a cell(e.g., a cancer cell, a macrophage, a bacterium, a fungus, a virus, or aparasite). A target is intended to be detected, diagnosed, impaired,destroyed or altered (e.g., functionally complemented) by the methodsprovided herein. The specific target may occur free or is associatedwith other entities in the circulatory system of a subject.

A “target self-antibody,” as used herein, is a self-antibody associatedwith an autoimmune disease. Such self-antibodies may be detected andanalyzed using antibody binding tests involving contacting the subject'santibodies to samples of the subject's own tissue, usually thyroid,stomach, liver, and kidney tissue. Antibodies binding to the “self”tissue (comprising self-antigens) indicate an autoimmune disorder.

“Transgene” or “exogenous nucleic acid” refers to a foreign or nativenucleotide sequence that is introduced into a syntheticmembrane-receiver complex. Transgene and exogenous nucleic acid are usedinterchangeably herein and encompass recombinant nucleic acids.

As used herein, “treat,” “treating,” and/or “treatment” are an approachfor obtaining beneficial or desired clinical results, pharmacologicand/or physiologic effect, e.g., alleviation of the symptoms, preventingor eliminating said symptoms, and refer to both therapeutic treatmentand prophylactic or preventative treatment of the specific disease,disorder or condition. Beneficial or desired clinical results,pharmacologic and/or physiologic effect include, but are not limited to,preventing the disease, disorder or condition from occurring in asubject that may be predisposed to the disease, disorder or conditionbut does not yet experience or exhibit symptoms of the disease(prophylactic treatment), alleviation of symptoms of the disease,disorder or condition, diminishment of extent of the disease, disorderor condition, stabilization (i.e., not worsening) of the disease,disorder or condition, preventing spread of the disease, disorder orcondition, delaying or slowing of the disease, disorder or conditionprogression, amelioration or palliation of the disease, disorder orcondition, and combinations thereof, as well as prolonging survival ascompared to expected survival if not receiving treatment.

A “therapeutic agent” or “therapeutic molecule” includes a compound ormolecule that, when present in an effective amount, produces a desiredtherapeutic effect, pharmacologic and/or physiologic effect on a subjectin need thereof.

The term “therapeutically effective amount” or “effective amount” is anamount of an agent being administered to a subject sufficient to effectbeneficial or desired clinical results, pharmacologic and/or physiologiceffects. An effective amount can be administered in one or moreadministrations. An effective amount is typically sufficient topalliate, ameliorate, stabilize, reverse, slow or delay the progressionof the disease state. The effective amount thus refers to a quantity ofan agent or frequency of administration of a specific quantity of anagent sufficient to reasonably achieve a desired therapeutic and/orprophylactic effect. For example, it may include an amount that resultsin the prevention of, treatment of, or a decrease in, the symptomsassociated with a disease or condition that is being treated, e.g., thediseases or medical conditions associated with a target polypeptide. Theamount of a therapeutic composition administered to the subject willdepend on the type and severity of the disease and on thecharacteristics of the individual, such as general health, pathologicconditions, diets, age, sex, body weight and tolerance to drugs. It willalso depend on the degree, severity and type of disease. Further, theeffective amount will depend on the methods of formulation andadministration used, e.g., administration time, administration route,excretion speed, and reaction sensitivity. The skilled artisan will beable to determine appropriate dosages depending on these and otherfactors. The compositions can also be administered in combination withone or more additional therapeutic compounds. A desirable dosage of thepharmaceutical composition may be in the range of about 0.001 to 100mg/kg for an adult. In one example, an intravenous administration isinitiated at a dose which is minimally effective, and the dose isincreased over a pre-selected time course until a positive effect isobserved. Subsequently, incremental increases in dosage are madelimiting to levels that produce a corresponding increase in effect whiletaking into account any adverse affects that may appear. Non-limitedexamples of suitable dosages can range, for example, from 1×10¹⁰ to1×10¹⁴, from 1×10¹¹ to 1×10¹³, or from 5×10¹¹ to 5×10¹² syntheticmembrane-receiver polypeptide complexes of the present invention.Specific examples include about 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰,1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹,1×10¹², or more synthetic membrane-receiver polypeptide complexes of thepresent invention. Each dose of synthetic membrane-receiver polypeptidecomplexes can be administered at intervals such as once daily, onceweekly, twice weekly, once monthly, or twice monthly.

“Unbound” refers to the state of a target with which the receiver iscapable of interacting. An unbound target is not associated with anotherentity or a receiver. An unbound receiver is not associated with anotherentity or a target. A target is considered “bound” once it is associatedwith the receiver or another entity. Unbound targets include solubleforms of the target in circulation. Bound targets include targets thatare embedded, associated with, linked to, or otherwise interacting withentities in circulation or peripheral tissue. Entities with which atarget may interact include circulating cells, peripheral endothelialtissue, immune complexes, glycolipids, microbes, immunoglobulins, serumalbumin, clotting factors, lipoproteins, and electrolytes.

A “variant” is a polypeptide which differs from the original protein byone or more amino acid substitutions, deletions, insertions, or othermodifications. These modifications do not significantly change thebiological activity of the original protein. In many cases, a variantretains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or100% of the biological activity of original protein. The biologicalactivity of a variant can also be higher than that of the originalprotein. A variant can be naturally-occurring, such as by allelicvariation or polymorphism, or be deliberately engineered.

The amino acid sequence of a variant is substantially identical to thatof the original protein. In many embodiments, a variant shares at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more global sequence identityor similarity with the original protein. Sequence identity or similaritycan be determined using various methods known in the art, such as BasicLocal Alignment Tool (BLAST), dot matrix analysis, or the dynamicprogramming method. In one example, the sequence identity or similarityis determined by using the Genetics Computer Group (GCG) programs GAP(Needleman-Wunsch algorithm). The amino acid sequences of a variant andthe original protein can be substantially identical in one or moreregions, but divergent in other regions.

As used herein, the term “vector” is a nucleic acid molecule, preferablyself-replicating, which transfers and/or replicates an inserted nucleicacid molecule, such as a transgene or exogenous nucleic acid into and/orbetween host cells. It includes a plasmid or viral chromosome into whosegenome a fragment of recombinant DNA is inserted and used to introducerecombinant DNA, or a transgene, into a synthetic membrane-receiverpolypeptide complex.

The “volume of distribution” (VD) is a pharmacological, theoreticalvolume that the total amount of administered drug would have to occupy(if it were uniformly distributed), to provide the same concentration asit is in blood plasma. A VD greater than the blood plasma indicates thatan agent is distributed in tissue in the rest of the body. The VD isinfluenced by solubility, charge, size, etc. Generally, non-polar agentswith high lipid solubility, agents with low rates of ionization or lowplasma binding capabilities have higher volumes of distribution thanagents that are more polar, more highly ionized or exhibit high plasmabinding. The volume of distribution is given by the following equation:V_(D)=total amount of drug in the body/drug blood plasma concentration.The units for Volume of Distribution are typically reported in (ml orliter)/kg body weight. A volume of distribution “equal to plasma volume”is relative to the volume of the circulatory system exclusive ofcirculating cells.

Synthetic Membrane-Receiver Complexes

Provided herein are synthetic membrane-receiver complexes, populations,pharmaceutical compositions, and dosage forms thereof, as well asmedical devices and kits comprising a formulation of the syntheticmembrane-receiver complexes.

The synthetic membrane-receiver complexes described herein comprise areceiver (e.g., a polypeptide) that is capable of interacting with atarget and further comprise a membrane comprising a polypeptide that isnot the receiver. The synthetic membrane-receiver complex has catalyticactivity independent of the receiver. Optionally, the syntheticmembrane-receiver complexes comprise a payload, for example atherapeutic agent.

In some embodiments, synthetic membrane-receiver complex are generatedusing cells as a source material. In certain embodiments, generating asynthetic membrane-receiver complex comprises the step of contacting anerythroid cell and platelets with a receiver. In certain embodiments,generating a synthetic membrane-receiver complex comprises the step ofcontacting a cell derived from a hematopoietic stell cell with areceiver.

In certain embodiments, synthetic membrane-receiver complexes areadministered, e.g., intravenously to the circulatory system of amammalian subject, such as a human. In some embodiments, themembrane-receiver complexes provide a natural barrier between a receiverand optionally a payload (e.g., therapeutic agent) and the immunesystem. In some embodiments, the synthetic membrane-receiver complexesare capable of residing in the circulatory system of a subject for anextended period of time allowing delivery of a therapeutic effect for alonger period of time than what can be achieved by delivery throughother methods currently used.

Synthetic membrane-receiver complexes may interact with a target in thecirculatory system of the subject. In some embodiments, theconcentration of an unbound target or total target in the circulatorysystem of the subject is reduced subsequent to its interaction with thereceiver exhibited in or on the synthetic membrane-receiver complex. Incertain embodiments, the presence or elevated concentration of a targetin circulation is associated with a disease, disorder or condition andreducing the concentration of the target leads to a reduction in diseaseburden, may alleviate a symptom of the disease or has some othertreatment effect. In some embodiments, a reduction in the concentrationof the target prevents the onset of a disease, disorder or condition.

Biodistribution is a substantial hurdle in drug delivery and efficacy.After a drug enters the systemic circulation, it is distributed to thebody's tissues. Distribution is generally uneven because of differencesin blood perfusion, tissue binding (e.g., because of lipid content),regional pH, and permeability of cell membranes. The entry rate of adrug into a tissue depends on the rate of blood flow to the tissue,tissue mass, and partition characteristics between blood and tissue.Distribution equilibrium (when entry and exit rates are the same)between blood and tissue is reached more rapidly in richly vascularizedareas, unless diffusion across cell membranes is the rate-limiting step.After equilibrium, drug concentrations in tissues and in extracellularfluids are reflected by the plasma concentration. Metabolism andexcretion occur simultaneously with distribution, making the processdynamic and complex.

The synthetic membrane-receiver complexes when formulated in apharmaceutical compositions suitable for administration into thecirculatory system of a subject can have a volume of distribution equalto the plasma volume of the subject. Advantages of the volume ofdistribution characteristic of the synthetic membrane-receiver complexesinclude that the biodistribution of the receiver when administered as asynthetic membrane-receiver complex into the circulatory system of asubject may be accurately predicted and/or that potential adverseextravascular effects of the receiver (e.g., an inflammatory response,an immune response, toxicity, etc.) are substantially reduced.

Distribution of a therapeutic composition out of the bloodstream andinto surrounding tissue increases the apparent volume of distribution tobe greater than the plasma volume of the subject. Therapeuticcompositions that exit the bloodstream and interact with surroundingtissue, e.g., adipose tissue or muscle, may interact with those tissuesin unpredictable ways and trigger adverse events. A therapeuticcomposition, such as a composition comprising a syntheticmembrane-receiver complex described herein, whose volume of distributiondoes not substantially exceed the plasma volume of the subject typicallyhas a safety profile that is superior to a therapeutic composition witha large volume of distribution. Further, the amount of a therapeuticcomposition that must be loaded to be effective (the effective amount)is in part dependent on the bioavailability of the therapeuticcomposition. Bioavailability is related to the composition's profile andrate of distribution into extra-vascular tissues, and thus its volume ofdistribution. By maintaining a precise and predictable volume ofdistribution, typically a therapeutic composition, such as a compositioncomprising a synthetic membrane-receiver complex described herein, willhave a more precise and predictable dose-effect relationship than atherapeutic composition with a less precise and predictable volume ofdistribution.

For example, the drug distribution rate for interstitial fluids of mosttissues is determined primarily by perfusion. For poorly perfusedtissues (e.g., muscle, fat), distribution is very slow, especially ifthe tissue has a high affinity for the drug. Endothelial cells liningthe vessel wall are connected by adherens, tight and gap junctions.These junctional complexes are related to those found at epithelialjunctions but with notable changes in terms of specific molecules andorganization. Endothelial junctional proteins play important roles intissue integrity but also in vascular permeability, leukocyteextravasation and angiogenesis. Small molecules, protein therapeutics,and viruses measure 1-30 nm and are capable of diffusing far beyond thevasculature based on lipophilicity, ability to bind plasma proteins, andcharge. A drug that is confined to the vasculature has a lesser volumeof tissue to occupy and thus may remain at an effective, therapeuticconcentration. In addition, the drug is unable to interact withperipheral tissues and potential off-target toxicity effects arelimited. Larger circulatory agents (e.g., between 1 micron and 20microns) do not pass through endothelial tight junctions which are lessthan 100 nm in width and endothelial cells are incapable of facilitatingthe transcytosis of agents of that size. In some embodiments, thesynthetic membrane-receiver complexes described herein measure between 1micon and 20 microns. The vascular properties of these agents limittheir diffusive capabilities to the bloodstream and concentrate thetherapeutic effect of any receiver or payload.

The synthetic membrane-receiver complexes described herein, in someembodiments, exhibit advantageous clearance properties. In someembodiments, synthetic membrane-receiver complexes may be degraded usinga natural degradation process, through the reticulo-endothelial system.Such degradation typically does not cause any or little side effects. Insome embodiments, receivers displayed on the synthetic membrane-receivercomplexes can be selectively trapped by organs of thereticulo-endothelial system.

The synthetic membrane-receiver complexes described herein are, in someembodiments, incapable of self-replication. In some embodiments, thesynthetic membrane-receiver complexes do not contain self-replicatingnucleic acids. Thus, such complexes do not carry a risk of uncontrolledcellular division, undesired protein expression and/or the potential oftriggering cytokine release syndrome.

Membrane Compositions of the Synthetic Membrane-Receiver Complexes

1. Lipids

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a membrane that has a mass of approximately 1×10̂-12 g and adensity of approximately 1.15 g/cm̂3. The mass of the membrane componentcan be assessed by separating it from the remainder of the complex usinghypotonic solutions of mildly alkaline buffer, see e.g., protocols inDodge et al 1963, Arch Biochem Biophys 100:119.

The synthetic membrane-receiver complex comprises a membrane. In someembodiments, the membrane comprises phosphatidylcholine, sphingomyelin,lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, or phosphatidic acid. In some embodiments, themembrane is a cell membrane.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises lipid molecules of the class of choline phospholipids, acidicphospholipids, and phosphatidylethanolamine.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises phosphatidylcholine, sphingomyelin, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, orphosphatidic acid.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises choline phospholipids in an approximate amount of 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%relative to the total lipid content of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises acidic phospholipids in an approximate amount of 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%relative to the total lipid content of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises phosphatidylcholine in an amount greater than 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%. 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or greater than 50%relative to the total lipid content of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises sphingomyelin in an amount greater than 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%. 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or greater than 50%relative to the total lipid content of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises lysophosphatidylcholine in an amount greater than 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, or greater than 10% relative to the total lipidcontent of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises phosphatidylethanolamine in an amount greater than 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%. 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or greater than50% relative to the total lipid content of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises phosphatidylserine in an amount greater than 1%, 1.5%, 2%,2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%. 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, or greater than 50% relative to the totallipid content of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises phosphatidylinositol in an amount greater than 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, or greater than 10% relative to the total lipidcontent of the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises phosphatidic acid in an amount greater than 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or greater than 10% relative to the total lipid contentof the complex.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises molecules from at least one, two, or three, of the followingclasses of molecules, including, but not limited to, cholinephospholipids, acidic phospholipids, and phosphatidylethanolamine.

In one embodiment the molar ratio of choline phospholipids to acidicphospholipids in the synthetic membrane-receiver polypeptide complex isless than 1:1000, approximately 1:1000, approximately 1:500,approximately 1:250, approximately 1:100, approximately 1:50,approximately 1:25, approximately 1:10, approximately 1:9, approximately1:8, approximately 1:7, approximately 1:6, approximately 1:5,approximately 1:4, approximately 1:3, approximately 1:2, approximately1:1, approximately 2:1, approximately 3:1, approximately 4:1,approximately 5:1, approximately 6:1, approximately 7:1, approximately8:1, approximately 9:1, approximately 10:1, approximately 25:1,approximately 50:1, approximately 100:1, approximately 250:1,approximately 500:1, approximately 1000:1, or greater than approximately1000:1.

In one embodiment the molar ratio of choline phospholipids tophosphatidyl ethanolamine in the synthetic membrane-receiver polypeptidecomplex is less than 1:1000, approximately 1:1000, approximately 1:500,approximately 1:250, approximately 1:100, approximately 1:50,approximately 1:25, approximately 1:10, approximately 1:9, approximately1:8, approximately 1:7, approximately 1:6, approximately 1:5,approximately 1:4, approximately 1:3, approximately 1:2, approximately1:1, approximately 2:1, approximately 3:1, approximately 4:1,approximately 5:1, approximately 6:1, approximately 7:1, approximately8:1, approximately 9:1, approximately 10:1, approximately 25:1,approximately 50:1, approximately 100:1, approximately 250:1,approximately 500:1, approximately 1000:1, or greater than approximately1000:1.

In one embodiment the molar ratio of phosphatidylethanolamine to acidicphospholipids in the synthetic membrane-receiver polypeptide complex isless than 1:1000, approximately 1:1000, approximately 1:500,approximately 1:250, approximately 1:100, approximately 1:50,approximately 1:25, approximately 1:10, approximately 1:9, approximately1:8, approximately 1:7, approximately 1:6, approximately 1:5,approximately 1:4, approximately 1:3, approximately 1:2, approximately1:1, approximately 2:1, approximately 3:1, approximately 4:1,approximately 5:1, approximately 6:1, approximately 7:1, approximately8:1, approximately 9:1, approximately 10:1, approximately 25:1,approximately 50:1, approximately 100:1, approximately 250:1,approximately 500:1, approximately 1000:1, or greater than approximately1000:1.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises molecules from at least one, two, three, four, five, six, orseven of the following classes of molecules, including, but not limitedto, phosphatidylcholine, sphingomyelin, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, orphosphatidic acid.

The lipid composition of the synthetic membrane-receiver polypeptidecomplex can be experimentally measured using methods known in the artincluding, e.g., gas-liquid chromatography or thin layer chromatography,see for example Dodge & Phillips, J Lipid Res 1967 8:667.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a lipid bilayer composed of an inner leaflet and an outerleaflet. The composition of the inner and outer leaflet can bedetermined by transbilayer distribution assays known in the art, seee.g., Kuypers et al. Biohim Biophys Acta 1985 819:170. In oneembodiment, the composition of the outer leaflet is betweenapproximately 70-90% choline phospholipids, between approximately 0-15%acidic phospholipids, and between approximately 5-30%phosphatidylethanolamine. In one embodiment, the composition of theinner leaflet is between approximately 15-40% choline phospholipids,between approximately 10-50% acidic phospholipids, and betweenapproximately 30-60% phosphatidylethanolamine.

2. Cholesterol

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises cholesterol. In one embodiment the cholesterol content isbetween approximately 3.0-5.5 nmol cholesterol per 10̂7 complexes. In oneembodiment, the cholesterol content is between approximately 1.8-3.5nmol cholesterol per 10̂7 complexes. In one embodiment the molar ratio ofcholesterol to phospholipids in the complex is between approximately0.5-1.5. In a preferred embodiment the molar ratio of cholesterol tophospholipids is between approximately 0.8-1.2. In a preferredembodiment the molar ratio of cholesterol to phospholipids is betweenapproximately 0.84-0.9. In a preferred embodiment the molar ratio ofcholesterol to phospholipids is between approximately 0.5-0.75. In apreferred embodiment the molar ratio of cholesterol to phospholipids isbetween approximately 0.55-0.6.

3. Lipids, Proteins, and Carbohydrates

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises polypeptides other than the receiver polypeptide. In oneembodiment, approximately 52% of the membrane mass is protein,approximately 40% is lipid, and approximately 8% is carbohydrate. In oneembodiment, approximately 7% of the carbohydrate content is comprised ofglycosphingolipids and approximately 93% of the carbohydrate content iscomprised of O-linked and N-linked oligosaccharides onmembrane-associated polypeptides.

In one embodiment the mass ratio of lipid to protein in the syntheticmembrane-receiver polypeptide complex is less than 1:1000, approximately1:1000, approximately 1:500, approximately 1:250, approximately 1:100,approximately 1:50, approximately 1:25, approximately 1:10,approximately 1:9, approximately 1:8, approximately 1:7, approximately1:6, approximately 1:5, approximately 1:4, approximately 1:3,approximately 1:2, approximately 1:1, approximately 2:1, approximately3:1, approximately 4:1, approximately 5:1, approximately 6:1,approximately 7:1, approximately 8:1, approximately 9:1, approximately10:1, approximately 25:1, approximately 50:1, approximately 100:1,approximately 250:1, approximately 500:1, approximately 1000:1, orgreater than approximately 1000:1.

In one embodiment the mass ratio of lipid to carbohydrate in thesynthetic membrane-receiver polypeptide complex is less than 1:1000,approximately 1:1000, approximately 1:500, approximately 1:250,approximately 1:100, approximately 1:50, approximately 1:25,approximately 1:10, approximately 1:9, approximately 1:8, approximately1:7, approximately 1:6, approximately 1:5, approximately 1:4,approximately 1:3, approximately 1:2, approximately 1:1, approximately2:1, approximately 3:1, approximately 4:1, approximately 5:1,approximately 6:1, approximately 7:1, approximately 8:1, approximately9:1, approximately 10:1, approximately 25:1, approximately 50:1,approximately 100:1, approximately 250:1, approximately 500:1,approximately 1000:1, or greater than approximately 1000:1.

In one embodiment the mass ratio of carbohydrate to protein in thesynthetic membrane-receiver polypeptide complex is less than 1:1000,approximately 1:1000, approximately 1:500, approximately 1:250,approximately 1:100, approximately 1:50, approximately 1:25,approximately 1:10, approximately 1:9, approximately 1:8, approximately1:7, approximately 1:6, approximately 1:5, approximately 1:4,approximately 1:3, approximately 1:2, approximately 1:1, approximately2:1, approximately 3:1, approximately 4:1, approximately 5:1,approximately 6:1, approximately 7:1, approximately 8:1, approximately9:1, approximately 10:1, approximately 25:1, approximately 50:1,approximately 100:1, approximately 250:1, approximately 500:1,approximately 1000:1, or greater than approximately 1000:1.

In one embodiment the area occupancy of protein in the syntheticmembrane-receiver polypeptide complex is approximately 23% and the areaoccupancy of lipid in the synthetic membrane-receiver polypeptidecomplex is approximately 77%.

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises a polypeptide selected from the following list, including butnot limited to, spectrin, myosin-like polypeptide, band 3, SLC4A1,actin, actin-like polypeptide, glyceraldehyde 3-P dehydrogenase (G3PD).

In one embodiment the synthetic membrane-receiver polypeptide complexcomprises at least one, two, three, four, five, six, or seven of thepolypeptides selected from the following list, including but not limitedto, spectrin, myosin-like polypeptide, band 3, SLC4A1, actin, actin-likepolypeptide, glyceraldehyde 3-P dehydrogenase (G3PD).

4. Additional Polypeptides

In some embodiments, the synthetic membrane-receiver complex comprisesat least one polypeptide that is not the receiver. In some embodiments,the synthetic membrane-receiver complex comprises at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine or at least ten polypeptides that are not thereceiver. In certain instances, the polypeptide is capable of anenzymatic or catalytic function independent of the receiver. Thenon-receiver polypeptide may be associated with the membrane of thesynthetic membrane-receiver complex.

In some embodiments, the non-receiver polypeptide may, e.g., stabilizethe synthetic membrane-receiver complex, target the syntheticmembrane-receiver complex to particular cells and tissues, engage thereticulo-endothelial system, protect the synthetic membrane-receivercomplex from macrophages and other phagocytic cells, and/or evade othercomponents of the innate immune system. Suitable polypeptides include,e.g., complement regulatory polypeptides, inhibitors of cell-mediateddegradation (e.g., CD47, CD55, and CD59), and anti-inflammatorypolypeptides. Alternatively or in addition, non-receiver polypeptidesmay shorten or control the half-life of the complex, including targetingto macrophages or other phagocytic cells. Suitable non-receiverpolypeptides may promote apoptosis or otherwise trigger opsonization. Insome embodiments, non-receiver polypeptides include polypeptidecarriers, pumps, and channels; Glut1, Band3, aquaporin 1, RhAH, NA/KATPase, Ca ATPase, Na—H exchanger, KCa3.1, KCl cotransporter, andcoenzyme Q10.

As many drugs are systemically delivered to the blood circulatorysystem, the answer to the problem of effective drug delivery oftenfocuses on maintaining the drug in the blood for extended periods oftime. Thus, the development of long-circulating (long half-life)therapeutics that remain biologically available in the blood forextended time periods is an unmet need. The synthetic membrane-receivercomplexes described herein can be modified to increase or decrease theirhalf-life in circulation. In some embodiments, the half-life of thereceiver and optionally the payload in circulation may be modified byaltering the half-life of the synthetic membrane-receiver complex. Insome instances, the half-life is increased and the increase may be, forinstance from about 1.5-fold to 20-fold increase in serum half-life.

In some embodiments, receivers may reside in circulation and may remainfunctional and active for substantially the duration of the syntheticmembrane-receiver complex in circulation. In some embodiments, receiversmay reside in circulation and may remain functional and active for morethan 21 days in circulation. In some instances, syntheticmembrane-receiver complexes and receivers may reside in circulation for30 days, 45 days, 60 days, 100 days, 120 days, or longer. In otherembodiments, the synthetic membrane-receiver complexes and receivers mayreside in circulation for several hours to several days, such as 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 days. Residency in the circulatory system, incertain embodiments, is determined by the presence or absence of certainpolypeptides on the synthetic membrane-receiver complex. For example,the synthetic membrane-receiver complex may comprise a CD47, CD55, orCD59 polypeptide or a functional fragment thereof.

CD47 is a membrane protein that interacts with the myeloid inhibitoryimmunoreceptor SIRPα (also termed CD172a or SHPS-1) that is present,e.g., on macrophages. Engagement of SIRPα by CD47 provides adown-regulatory signal that inhibits host cell phagocytosis. Forexample, high levels of CD47 allow cancer cells to avoid phagocytosisdespite the presence pro-phagocytic signals, such as high levels ofcalreticulin. CD47 also has further roles in cell adhesion, e.g., byacting as an adhesion receptor for THBS1 on platelets and in themodulation of integrins. CD47 interaction with SIRPα further preventsmaturation of immature dendritic cells, inhibits cytokine production bymature dendritic cells. CD47 interaction with SIRPγ mediates cell-celladhesion, enhances superantigen-dependent T-cell-mediated proliferationand co-stimulates T-cell activation.

CD47 is a 50 kDa membrane receptor that has extracellular N-terminal IgVdomain, five transmembrane domains, and a short C-terminal intracellulartail. There are four alternatively spliced isoforms of CD47 that differonly in the length of their cytoplasmic tail. In some embodiments, thesynthetic membrane-receiver complex may comprise a CD47 or a functionalfragment thereof comprising one or more of: the extracellular N-terminalIgV domain, one, two, three, four, or five transmembrane domains, and/orthe short C-terminal intracellular tail. The cytoplasmic tail can befound as four different splice isoforms ranging from 4 to 36 aminoacids. The 16 amino acid form 2 is expressed in all cells ofhematopoietic origin and in endothelial and epithelial cells. The 36amino acid form 4 is expressed primarily in neurons, intestine, andtestis. The 4 amino acid form 1 is found in epithelial and endothelialcells. The expression pattern of the 23 amino acid form 3 resembles thatof form 4. In some embodiments, the synthetic membrane-receiver complexcomprises CD47 or a functional fragment thereof that is of one of form1, from 2, form 3, or from 4. In some embodiments, the syntheticmembrane-receiver complex does not comprise form 2. In some embodiments,the synthetic membrane-receiver complex comprises CD47 polypeptide or afunctional polypeptide fragment thereof in an amount or copy numbersufficient to reside in circulation for 15 days, 21 days, 30 days, 45days, 60 days, 100 days, 120 days, or longer. In some embodiments, thesynthetic membrane-receiver complex comprises a modified CD47, such as aconformational change. For example, a conformational change in CD47 isintroduced so that the modified CD47 is capable of interacting withTSP-1. In one embodiment, the modified CD47 comprising theconformational change creates a different binding site for SIRPα. Insome embodiments, the synthetic membrane-receiver complex comprises amodified CD47 polypeptide or a functional polypeptide fragment thereofcomprising a conformational change in an amount or copy numbersufficient to reside in circulation for less than 10, 9, 8, 7, 6, 5, 4,3, 2, or less than 1 day. In certain embodiments, the syntheticmembrane-receiver complex comprises a fusion of a CD47 isoform to theextracellular domain of a native erythroid polypeptide. For example, theN terminus of glycophorin A may be fused to the CD47 polypeptide orfunctional fragment thereof, which may lead to a reduction of theSIRPα-mediated signal to macrophages to phagocytose the syntheticmembrane-receiver complex.

In some embodiments, generating synthetic membrane-receiver complexesincludes the step of contacting a receiver (e.g., a polypeptide) with acell, such as an erythroid cell or a platelet. CD47 is expressed inerythrocytes and platelets to mediate phagocytosis. In some embodiments,the natural levels of CD47 are altered in erythrocytes or platelets,e.g., by over-expression or inhibition of CD47 expression using anysuitable method, such as the introduction of exogenous nucleic acids(e.g., expression vectors, CD47 mRNA, CD47 siRNA, and the like). In someembodiments, the natural levels of CD47 are altered such that thesynthetic membrane-receiver complex resides in circulation for 15 days,21 days, 30 days, 45 days, 60 days, 100 days, 120 days, or longer. Insome embodiments, the natural levels of CD47 are altered such that thesynthetic membrane-receiver complex resides in circulation for less than10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day.

For example, synthetic membrane-receiver complexes that are administeredto a subject may comprise elevated CD47 levels when compared to nativelevels of a suitable control. Elevated CD47 levels may be achieved,e.g., by exogenous expression by the synthetic membrane-receiver complexof CD47 from an exogenous nucleic acid, by loading of CD47 mRNA into thecomplex, or by conjugating CD47 polypeptide to the surface of thecomplex. Elevated CD47 levels are useful to increase the half-life ofthe population of synthetic membrane-receiver complexes in thecirculatory system of the subject. The synthetic membrane-receivercomplexes comprise a receiver and optionally a payload, such as atherapeutic agent. In some embodiments, increasing the half-life of thesynthetic membrane-receiver complex increases the half-life of thereceiver and/or the optional payload in circulation, thereby potentiallyincreasing the therapeutic window in which the receiver and/or payloadis active. In one instance, a population of 10¹¹ syntheticmembrane-receiver polypeptide complexes comprises an adenosine deaminasereceiver and an exogenous CD47 polypeptide on its surface. Whenadministered to a subject with an enzyme deficiency, such as ADA-SCID,the half-life of the synthetic membrane-receiver polypeptide complex isextended beyond that of a complex not comprising exogenous CD47polypeptide and the subject requires less frequent dosing. Half-lifeextension is a particular advantage when compared to current enzymetherapies not involving synthetic membrane-receiver polypeptidecomplexes.

In some embodiments, CD47 is altered by heparin and/or chondroitinsulfate glycosaminoglycan (GAG) chains. In some embodiments, thesynthetic membrane-receiver complex expresses CD47 as a proteoglycan. Insome embodiments, the synthetic membrane-receiver complex comprises aCD47 proteoglycan that is conjugated to the complex. In one embodiment,the CD47 proteoglycan comprises heparin and/or chondroitin sulfateglycosaminoglycan (GAG) chains. In one embodiment, that CD47proteoglycan has a size of greater than 150 kDa, 200 kDa, or greaterthan 250 kDa. In one embodiment, CD47 comprises one or more GAG chainsat Ser64.

In some embodiments, the residency of a synthetic membrane-receivercomplex, e.g., generated using erythroid cells or platelets can befurther modulated by changing the amount or number of oxidized lipids onthe membrane of the synthetic membrane-receiver complex. In oneembodiment, the synthetic membrane-receiver complex comprises oxidizedlipids in an amount effective to reside in circulation for less than 10,9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day. In one embodiment, thesynthetic membrane-receiver complex comprises oxidized lipids in anamount effective to reside in circulation for 15 days, 21 days, 30 days,45 days, 60 days, 100 days, 120 days, or longer. In some embodiments,the amount of oxidized lipids in the membrane are altered such thatmobility of CD47 is increased or decreased, thereby aiding or hindering,respectively the ability of CD47 to cluster on the membrane. (See,Olsson, Department of Integrative Medical Biology, Section for Histologyand Cell Biology, Umea University, Umea, Sweden, 2008).

CD55, also known as complement decay-accelerating factor or DAF, is a 70kDa membrane protein. CD55 recognizes C4b and C3b fragments of thecomplement system that are created during C4 (classical complementpathway and lectin pathway) and C3 (alternate complement pathway)activation. It is thought that interaction of CD55 with cell-associatedC4b and C3b proteins interferes with their ability to catalyze theconversion of C2 and factor B to active C2a and Bb and thereby preventsthe formation of C4b2a and C3bBb, the amplification convertases of thecomplement cascade. CD55 is thought to block the formation of membraneattack complexes. CD55 may prevent lysis by the complement cascade. Insome embodiments, the synthetic membrane-receiver complex comprises CD55polypeptide or a functional polypeptide fragment thereof in an amount orcopy number sufficient to reside in circulation for 15 days, 21 days, 30days, 45 days, 60 days, 100 days, 120 days, or longer. In someembodiments, the synthetic membrane-receiver complex comprises anexogenous CD55 polypeptide and an exogenous CD47 polypeptide orfunctional polypeptide fragments thereof in an amount, copy numberand/or ratio sufficient to reside in circulation for 15 days, 21 days,30 days, 45 days, 60 days, 100 days, 120 days, or longer.

CD59 glycoprotein also known as MAC-inhibitory protein (MAC-IP),membrane inhibitor of reactive lysis (MIRL), protectin, or HRF is aprotein that attaches to host cells via a glycophosphatidylinositol(GPI) anchor. When complement activation leads to deposition of C5b678on host cells, CD59 can prevent C9 from polymerizing and forming thecomplement membrane attack complex. CD59 may prevent lysis by thecomplement cascade. In some embodiments, the synthetic membrane-receivercomplex comprises CD59 polypeptide or a functional polypeptide fragmentthereof in an amount or copy number sufficient to reside in circulationfor 15 days, 21 days, 30 days, 45 days, 60 days, 100 days, 120 days, orlonger. In some embodiments, the synthetic membrane-receiver complexcomprises an exogenous CD59 polypeptide and an exogenous CD47polypeptide or functional polypeptide fragments thereof in an amount,copy number and/or ratio sufficient to reside in circulation for 15days, 21 days, 30 days, 45 days, 60 days, 100 days, 120 days, or longer.

In some embodiments, the synthetic membrane-receiver complex comprisesone or more of an exogenous CD55 polypeptide, an exogenous CD59polypeptide and/or an exogenous CD47 polypeptide or functionalpolypeptide fragments thereof in an amount, copy number and/or ratiosufficient to reside in circulation for 15 days, 21 days, 30 days, 45days, 60 days, 100 days, 120 days, or longer.

Effective amounts of CD47, CD55, and CD59 include 10², 10³, 10⁴, 10⁵,10⁶, 10⁷, 10⁹ polypeptides per synthetic membrane-receiver complex.Alternatively, an effective amount is the amount capable of extendingthe synthetic membrane-receiver polypeptide complex's half-life by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 400%, 800%, 1,000%,or 10,000% relative to the half-life that the syntheticmembrane-receiver polypeptide complex would exhibit without thepolypeptides.

Receivers

Provided herein are receivers that are exhibited by syntheticmembrane-receiver complexes. In some embodiments, a receiver is capableof interacting with a target, e.g., to associate with or bind to atarget. A receiver can comprise or may consist essentially of apolypeptide. In some embodiments, the receiver comprises a polypeptide,a carbohydrate, a nucleic acid, a lipid, a small molecule, or acombination thereof. In some embodiments receivers do not interact witha target but act as payloads to be delivered by the syntheticmembrane-receiver complex to a cell, tissue or other site in the body ofa subject.

In some embodiments, receivers comprise polypeptides. Receiverpolypeptides may range in size from 6 amino acids to 3000 amino acidsand may exceed 6, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,150, 200, 300, 400 or may exceed 500 amino acids. Receiver polypeptidesmay range in size from about 20 amino acids to about 500 amino acids,from about 30 amino acids to about 500 amino acids or from about 40amino acids to about 500 amino acids.

In some embodiments, the receiver polypeptide comprises a chimeric orfusion protein which may comprise two or more distinct protein domains.These chimeric receivers are heterologous or exogenous in the sense thatthe various domains are derived from different sources, and as such, arenot found together in nature and can be encoded e.g., by exogenousnucleic acids. Receiver polypeptides can be produced by a number ofmethods, many of which are well known in the art and also describedherein. For example, receiver polypeptides can be obtained by extraction(e.g., from isolated cells), by expression of an exogenous nucleic acidencoding the receiver polypeptide, or by chemical synthesis. Receiverpolypeptides can be produced by, for example, recombinant technology,and expression vectors encoding the polypeptide introduced into hostcells (e.g., by transformation or transfection) for expression of theencoded receiver polypeptide.

There are a variety of conservative changes that can generally be madeto an amino acid sequence without altering activity. These changes aretermed conservative substitutions or mutations; that is, an amino acidbelonging to a grouping of amino acids having a particular size, chargeor other characteristic can be substituted for another amino acid.Substitutions for an amino acid sequence may be selected from othermembers of the class to which the amino acid belongs. For example, thenonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, methionine, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations are not expected to substantially affectapparent molecular weight as determined by polyacrylamide gelelectrophoresis or isoelectric point. Conservative substitutions alsoinclude substituting optical isomers of the sequences for other opticalisomers, specifically D amino acids for L amino acids for one or moreresidues of a sequence. Moreover, all of the amino acids in a sequencemay undergo a D to L isomer substitution. Exemplary conservativesubstitutions include, but are not limited to, Lys for Arg and viceversa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free ˜OH ismaintained; and Gln for Asn to maintain a free NH₂. Moreover, pointmutations, deletions, and insertions of the polypeptide sequences orcorresponding nucleic acid sequences may in some cases be made without aloss of function of the polypeptide or nucleic acid fragment.Substitutions may include, e.g., 1, 2, 3, or more residues. Any teachingof a specific amino acid sequence or an exogenous nucleic acid encodingthe polypeptide or teaching of the name of the name thereof includes anyconservative substitution point mutations, deletions, and insertions ofthose polypeptide sequences or corresponding nucleic acid sequences andany sequence deposited for the protein or gene in a database that can bemade without a loss of function of the polypeptide or nucleic acidfragment.

In some embodiments, the receiver polypeptide is associated with themembrane of the synthetic membrane-receiver polypeptide complex. Inother embodiments, the receiver polypeptide is not associated with themembrane of the synthetic membrane-receiver polypeptide complex.

In one embodiment the mass ratio of lipid to receiver in the syntheticmembrane-receiver polypeptide complex is less than 1:1000, approximately1:1000, approximately 1:500, approximately 1:250, approximately 1:100,approximately 1:50, approximately 1:25, approximately 1:10,approximately 1:9, approximately 1:8, approximately 1:7, approximately1:6, approximately 1:5, approximately 1:4, approximately 1:3,approximately 1:2, approximately 1:1, approximately 2:1, approximately3:1, approximately 4:1, approximately 5:1, approximately 6:1,approximately 7:1, approximately 8:1, approximately 9:1, approximately10:1, approximately 25:1, approximately 50:1, approximately 100:1,approximately 250:1, approximately 500:1, approximately 1000:1,approximately 10,000:1, approximately 100,000:1, approximately1,000,000:1, approximately 10,000,000:1, approximately 100,000,000:1,approximately 1,000,000,000:1 or greater than approximately1,000,000,000:1.

In one embodiment the mass ratio of non-receiver polypeptide to receiverin the synthetic membrane-receiver polypeptide complex is less than1:1000, approximately 1:1000, approximately 1:500, approximately 1:250,approximately 1:100, approximately 1:50, approximately 1:25,approximately 1:10, approximately 1:9, approximately 1:8, approximately1:7, approximately 1:6, approximately 1:5, approximately 1:4,approximately 1:3, approximately 1:2, approximately 1:1, approximately2:1, approximately 3:1, approximately 4:1, approximately 5:1,approximately 6:1, approximately 7:1, approximately 8:1, approximately9:1, approximately 10:1, approximately 25:1, approximately 50:1,approximately 100:1, approximately 250:1, approximately 500:1,approximately 1000:1, approximately 10,000:1, approximately 100,000:1,approximately 1,000,000:1, approximately 10,000,000:1, approximately100,000,000:1, approximately 1,000,000,000:1 or greater thanapproximately 1,000,000,000:1.

In certain embodiments, the polypeptide receiver is located on thesurface and is exposed to the environment around the syntheticmembrane-receiver polypeptide complex. In some embodiments, thepolypeptide receiver is located inside and faces the unexposed side ofthe synthetic membrane-receiver polypeptide complex.

In certain embodiments, the polypeptide receiver comprises at least oneof the following domains, an S domain (surface), an A domain (anchor),and/or a U domain (unexposed), wherein the S domain is a surface domainexposed to the environment around the synthetic membrane-receiverpolypeptide complex, wherein the A domain is an anchor, and wherein theU domain is located within and/or faces the unexposed side of thesynthetic membrane-receiver polypeptide complex.

Optionally the receiver polypeptide comprises i) one or more additionalS domains, termed S′ domains, or ii) one or more additional U domains,termed U′ domains.

In some embodiments, the S domain and the A domain form part of the samepolypeptide chain.

In some embodiments, the A domain and the U domain form part of the samepolypeptide chain.

In some embodiments, any one or more of the S, A, U domain is added tothe synthetic membrane-receiver polypeptide complex externally.

In some embodiments, any one or more of the S, A, U domain is producedwithin the synthetic membrane-receiver polypeptide complex.

In some embodiments, any one or more of the S, A, U domain is apolypeptide.

In some embodiments, any one or more of the S, A, U domain is not apolypeptide.

Schematics of exemplary conformations of receivers within or onsynthetic membrane-receiver complexes are shown in FIGS. 14A, 14B, and14C.

1. The A Domain

In certain embodiments, the A domain is a membrane polypeptide. The Adomain can be, e.g., an integral membrane polypeptide or a membraneassociated polypeptide.

The A domain may be selected from one of the following classes,including but not limited to, for example, alpha-helical bitopic,alpha-helical polytopic, beta-barrel transmembrane, all alphamonotopic/peripheral, all beta monotopic/peripheral, alpha/betamonotopic/peripheral, alpha+beta monotopic/peripheral, alpha helicalpeptides, beta-hairpin peptides, beta-helical peptides, type 1transmembrane protein (N-terminus extracellular), type 2 transmembraneprotein (N-terminus intracellular), type 3 transmembrane protein, type4A transmembrane protein, type 4B transmembrane protein, lipid-anchoredprotein, glycosylphosphatidylinositol (GPI) anchored protein, prenylchain anchored protein, or peptides of nonregular structure.

In certain embodiments, the A domain is endogenous, e.g., endogenous toan erythroid cell, a platelet, or a hematopoietic cell. In someembodiments, the A domain is endogenous to a mammalian cell.

In certain embodiments, the A domain is exogenous, e.g., exogenous to anerythroid cell, a platelet, or a hematopoietic cell. In someembodiments, the A domain is exogenous to a mammalian cell.

The A domain may be selected from the the following molecules orfragments thereof, including but not limited to, CD1, CD2, CD3, CD4,CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD13, CD14,CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26,CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38,CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49a,CD49b, CD49c, CD49d, CD49e, CD49f, CD53, CD54, CD55, CD56, CD57, CD58,CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD73,CD74, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95,CD96, CD100, CD103, CD105, CD106, CD107, CD107a, CD107b, CD109, CD117,CD120, CD122, CD123, CD127, CD132, CD133, CD134, CD135, CD138, CD141,CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD155, CD156, CD158,CD163, CD165, CD166, CD168, CD184, CDw186, CD195, CD197, CDw199, CD209,CD202a, CD220, CD221, CD235a, CD271, CD279, CD303, CD304, CD309, CD326,Ras-Related protein 1A, semaporin 7A precursor, Calcium andintegrin-binding protein 1, 55 kDa erythrocyte membrane protein,Flotillin-1, Flotillin-2, Erythroid membrane-associated protein,eukaryotic translation initiation factor 2C 2, cytochrome b5 reductase,cell division control protein 42 homolog, KIAA1363 protein, band3,annexin VII, aquaporin, Ecto-ADP-ribosyltransferase 4, Kell, LFA-3,soulute carrier family 2 member 1, LGALS3 protein, Urea transporter, Rhblood CE group antigen polypeptide, Rh-associated glycoprotein, Dematin,ABO blood groups, Aquaporin 3, Aubergers, Band 3, Basigin, C41, CD44,Cis AB, Colton antigen, Complement Component 4, CR1, DAF, Diego, Duffy,Hh/Bombay antigen, ii antigen, Indian blood group, Kell, Kidd, Lewisantigen, Lutheran antigen, MNS antigen system, Cost group, Er group,Dematin, Stomatin, Tropomyosin, Glucose transporter, Adducin, Rabphilin,C1 tetrahydrofolate synthase, Vel group, Lan antigen, At antigen, Jrantigen, AnWj antigen, Sd antigen, Batty, Bilkes, Box, Christiansen,HJK, HOFM, JFV, JONEs, Jensen, Katagiri, Livesay, Milne, Oldeide,Peters, Rasmussen, Reid, REIT, SARA, Rhesus blood D group, Aldolase,Tropomodulin, Arginase, Creatine kinase, B-Cam protein, Rap 1A,Bennett-Goodspeed, P antigen system, Rh blood groupXg antigen system, XKprotein, Yt/Cartwright antigen system, CD58, Rh, Scianna, Radin, DARC(Duffy), CR1 Knops-McCoy, DAF Cromer, Gerbich (GYPC), CD47, GlycophorinA, Band 3 (AE3), GYPB Ss, C4A, C4B Chido, Rodgers C4 component ofcomplement, HLA Bg HLA class I, RHAG Rh-associated Ammonium transport,Glycoprotein, Colton (Co) Water channel protein, ACHE Cartwright (Yt)Acetylcholinesterase, Glutathione transferase, Glycophorin C, Aquaporin,Erythroblast associated membrane protein, CD44, Synaptobrevin 2,Ribonuclease, Duodenal cytochrome B, ABO glycosyl transferases, CD59,CD44 Indian (In), AnWj Adhesion receptor, MER2, DOK DombrockADP-ribosyltransferase, SEMA7A JMH Putative adhesion receptor, UMOD SdaTamm-Horsfall protein (uromodulin), Diego (Di), Wright (Wr) Anionchannel protein (band 3, AE1), Kidd (Jk) Urea transporter, FUT3 Lewis(Le) alpha(1,3) fucosyltransferase, OK Oka Neurothelin, putativeadhesion molecule, LW Adhesion receptor, FUT2 Secretor (Se) alpha(1,2)fucosyltransferase, FUT1 Hh alpha(1,2) fucosyltransferase, LU Lutheran(Lu) Adhesion receptor, P1 Glycosyltransferase, XK Kx Putativeneurotransmitter transporter, XG Xg formerly called PBDX, MIC2,Hemoglobin, Ankyrin, Spectrin, KEL Kell (forms K, k, Kp, Js)Metalloproteinase, Torkildsen antigen, coenzyme Q10, Rab 35, Ral Abinding protein, Zona pellucida binding protein, Lyn B protein, KIaa1741protein, DC38, Calcium transporting ATPase, GPIX, GPIba, GPIbb, GPV,GPIb-IX-V, GPVI, GPIa/IIa, GPIIb/IIIa, GPV/IIa.

2. The S Domain

In some embodiments, the S domain is a protein or a polypeptide. Inother embodiments, the S domain is a nucleic acid. In some embodiments,the S domain is a chemical. In certain embodiment the S domain is asmall molecule.

In some embodiments, the S domain is a polypeptide selected from orderived from one or more of the following classes, including but notlimited to, a flexible linker, an epitope tag, an enzyme, a protease, anuclease, a receiver, an antibody-like molecule, a ligand of anantibody, a growth factor, a cytokine, a chemokine, a growth factorreceptor, a cytokine receptor, a chemokine receptor, an enzymaticrecognition sequence, a transpeptidase recognition sequence, a proteaserecognition sequence, a cleavable domain, an intein, a DNA bindingprotein, and RNA binding protein, a complement regulatory molecule, acomplement cascade molecule, a clotting cascade molecule, a chelator, acomplement regulatory domain, an SCR domain, a CCP domain, animmunoglobulin or immunogloblulin-like domain, an armadillo repeat, aleucine zipper, a dealth effector domain, a cadherein repeat, an EFhand, a phosphotyrosine binding domain, a pleckstrin homology domain, anSCR homology 2 domain, a zinc finger domain, a cyclic peptide, acell-penetrating peptide.

In some embodiments, the S domain is a non-polypeptide molecule, forexample a nucleic acid, a carbohydrate, or a small molecule. In someembodiments, the S domain is a nucleic acid selected from one or more ofthe following classes, including but not limited to, a DNA aptamer, anRNA aptamer, an siRNA, a shRNA, a single-strand RNA probe, a singlestrand DNA probe, an mRNA, a chemically modified oligonucleotide. Insome embodiments, the S domain is a small molecule selected from one ormore of the following classes, including but not limited to, a chelator,DOTA, a radionuclide, an isotope, an imaging agent, a fluorescentmolecule, a chemiluminescent molecule, a gas.

3. The U Domain

In some embodiments, the U domain is a protein or a polypeptide. Inother embodiments, the U domain is a nucleic acid. In some embodiments,the U domain is a chemical. In certain embodiment the U domain is asmall molecule.

In some embodiments, the U domain is a polypeptide selected from orderived from one or more of the following classes, including but notlimited to, a flexible linker, an epitope tag, an enzyme, a protease, anuclease, a receiver, an antibody-like molecule, a ligand of anantibody, a growth factor, a cytokine, a chemokine, a growth factorreceptor, a cytokine receptor, a chemokine receptor, an enzymaticrecognition sequence, a transpeptidase recognition sequence, a proteaserecognition sequence, a cleavable domain, an intein, a DNA bindingprotein, and RNA binding protein, a complement regulatory molecule, acomplement cascade molecule, a clotting cascade molecule, a chelator, acomplement regulatory domain, an SCR domain, a CCP domain, animmunoglobulin or immunogloblulin-like domain, an armadillo repeat, aleucine zipper, a dealth effector domain, a cadherein repeat, an EFhand, a phosphotyrosine binding domain, a pleckstrin homology domain, anSCR homology 2 domain, a zinc finger domain, a cyclic peptide, acell-penetrating peptide, a kinase domain, aphosphatase domain, acytoskeletal protein, a protein that interacts with the cytoskeletalprotein, a G-protein coupled receptor, a tyrosine kinase, an ITIMdomain, an ITAM domain.

In some embodiments, the U domain is a non-polypeptide molecule, forexample a nucleic acid, a carbohydrate, or a small molecule. In someembodiments, the U domain is a nucleic acid selected from one or more ofthe following classes, including but not limited to, a DNA aptamer, anRNA aptamer, an siRNA, a shRNA, a single-strand RNA probe, a singlestrand DNA probe, an mRNA, a chemically modified oligonucleotide. Insome embodiments, the U domain is a small molecule selected from one ormore of the following classes, including but not limited to, a chelator,DOTA, a radionuclide, an isotope, an imaging agent, a fluorescentmolecule, a chemiluminescent molecule, a gas.

Examples of Receiver Polypeptides

Examples of receiver polypeptides include: the polypeptide receivercomprises glycophorin A with HA epitope tag at the N terminus; thepolypeptide receiver comprises the leader sequence of glycophorin A, HAepitope tag, and the body sequence of glycophorin A; the polypeptidereceiver comprises complement receptor 1 (CR1); the polypeptide receivercomprises the leader sequence of CR1, HA epitope tag, the body sequenceof CR1; the polypeptide receiver comprises the leader sequence of CR1,HA epitope tag, six SCR domains of LHR-A and LHR-B of CR1, the membraneproximal two SCR domains of CR1, the transmembrane region of CR1, andthe intracellular region of CR1; the polypeptide receiver comprises theleader sequence of CR1, HA epitope tag, nine SCR domains of LHR-A andLHR-B and LHR-C of CR1, the membrane proximal two SCR domains of CR1,the transmembrane region of CR1, and the intracellular region of CR1;the polypeptide receiver comprises the leader sequence of CR1, LHR-A ofCR1, LHR-B of CR1, LHR-C of CR1, the membrane proximal two SCR domainsof CR1, the transmembrane region of CR1, and the intracellular region ofCR1; the polypeptide receiver comprises leader sequence of CR1, LHR-A ofCR1, LHR-B of CR1, LHR-C of CR1, the membrane proximal two SCR domainsof CR1, the transmembrane region and intracellular region of glycophorinA; the polypeptide receiver comprises the leader sequence of glycophorinA, an antibody scFv against hepatitis B surface antigen (scFv), a(Gly3Ser)2 (SEQ ID NO: 23) flexible linker, HA epitope tag, and the bodyof glycophorin A; the polypeptide receiver comprises Kell, a (Gly3Ser)2(SEQ ID NO: 23) flexible linker, HA epitope tag, and scFv; thepolypeptide receiver comprises Kell and HA epitope tag; the polypeptidereceiver comprises a 71-amino acid N-terminal fragment of Kell and an HAepitope tag; the polypeptide receiver comprises a 71-amino acidN-terminal fragment of Kell, a (Gly3Ser)2 (SEQ ID NO: 23) flexiblelinker, and an HA epitope tag; the polypeptide receiver comprises a79-amino acid N-terminal fragment of Kell and an HA epitope tag; thepolypeptide receiver comprises a 79-amino acid N-terminal fragment ofKell, a (Gly3Ser)2 (SEQ ID NO: 23) flexible linker, and an HA epitopetag; the polypeptide receiver comprises a 71-amino acid N-terminalfragment of Kell, a (Gly3Ser)2 (SEQ ID NO: 23) flexible linker, scFv,and an HA epitope tag; the polypeptide receiver comprises a 79-aminoacid N-terminal fragment of Kell, a (Gly3Ser)2 (SEQ ID NO: 23) flexiblelinker, scFv, and an HA epitope tag; the polypeptide receiver comprisesthe leader sequence of CD55, scFv, an HA epitope tag, and the terminal37 amino acids of CD55; the polypeptide receiver comprises the leadersequence of CD55, an HA epitope tag, and the body of CD55. In oneembodiment, the polypeptide receiver comprises the leader sequence ofCD59, scFv, an HA epitope tag, and the body of CD59; the polypeptidereceiver comprises the leader sequence of CD59, and HA epitope tag, andthe body of CD59; the polypeptide receiver comprises adenosine deaminaseand an HA epitope tag; the polypeptide receiver comprises phenylalaninehydroxylase and an HA epitope tag; the polypeptide receiver comprisesadenosine deaminase, a (Gly3Ser)2 (SEQ ID NO: 23) flexible linker,phenylalanine hydroxylase, and an HA epitope tag; the polypeptidereceiver comprises glycophorin A, adenosine deaminase at the cytoplasmicC terminus, and an HA epitope tag; the polypeptide receiver comprisesglycophorin A, phenylalanine hydroxylase at the cytoplasmic C terminus,and an HA epitope tag.

In certain embodiments, the receiver is capable or interacting with amacrophage. The receiver polypeptide may comprise one or more of: thecomplement receptor (Rieu et al., J. Cell Biol. 127:2081-2091 (1994)),the scavenger receptor (Brasseur et al., Photochem. Photobiol.69:345-352 (1999)), the transferrin receptor (Dreier et al., Bioconjug.Chem. 9:482-489 (1998); Hamblin et al., J. Photochem. Photobiol. 26:4556(1994)); the Fc receptor (Rojanasakul et al., Pharm. Res. 11:1731-1733(1994)); and the mannose receptor (Frankel et al., Carbohydr. Res.300:251-258 (1997); Chakrabarty et al., J. Protozool. 37:358-364(1990)).

Other receivers capable or interacting with a macrophages include: lowdensity lipoproteins (Mankertz et al., Biochem. Biophys. Res. Commun.240:112-115 (1997); von Baeyer et al., Int. J. Clin. Pharmacol. Ther.Toxicol. 31:382-386 (1993)), very low density lipoproteins (Tabas etal., J. Cell Biol. 115:1547-1560 (1991)), mannose residues and othercarbohydrate moieties (Pittet et al., Nucl. Med. Biol. 22:355-365(1995)), poly-cationic molecules, such as poly-L-lysine (Hamblin et al.,J. Photochem. Photobiol. 26:45-56 (1994)), liposomes (Bakker-Woudenberget al., J. Drug Target. 2:363-371 (1994); Betageri et al., J. Pharm.Pharmacol. 45:48-53 (1993)) and 2-macroglobulin (Chu et al., J. Immunol.152:1538-1545 (1994)).

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising an extracellular domain of an HIVcoreceptor. In some embodiments, the synthetic membrane-receiver complexdoes not comprise a receiver capable of binding to a virus. In someembodiments, the synthetic membrane-receiver complex does not comprise areceiver comprising CD4. In some embodiments, the syntheticmembrane-receiver complex does not comprise a receiver comprising an HIVcoreceptor. In some embodiments, the synthetic membrane-receiver complexdoes not comprise a receiver comprising CXCR4, CCR5, CCR1, CCR2, CCR3,CCR4, CCR8, CXCR1, CXCR2, CXCR3, CXCR6, GPR15, APJ, CMKLR1, or CX3CR1 ora combination thereof.

In some embodiments, the synthetic membrane-receiver complex does notcontain an exogenous nucleic acid encoding an adenosine deaminasereceiver. In some embodiments, the synthetic membrane-receiver complexdoes not comprise a receiver comprising adenosine deaminase (ADA).

In some embodiments, the synthetic membrane-receiver complex does notcomprise an exogenous nucleic acid encoding an oncogene. In someembodiments, the synthetic membrane-receiver complex does not comprise areceiver comprising oncogene.

In some embodiments, the synthetic membrane-receiver complex does notcontain an exogenous nucleic acid encoding cdx1, cdx2, or cdx4. In someembodiments, the synthetic membrane-receiver complex does not comprise areceiver comprising cdx1, cdx2, or cdx4, or a combination thereof.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising a chimeric polypeptide comprising aligand binding domain. In some embodiments, the syntheticmembrane-receiver complex does not comprise a receiver comprising an Sdomain that is capable of binding a ligand. In some embodiments, thesynthetic membrane-receiver complex does not comprise a receivercomprising CD3ζ; CD3η, an IL-2 receptor, an IL-3 receptor, an IL-4receptor, an IL-7 receptor, an IL-11 receptor, an IL-13 receptor, aGM-CSF receptor, a LIF receptor, a CNTF receptor, an oncostatin Mreceptor, a TGF-β receptor, an EGF receptor, ATR2/neu, a HER2/neu, aHER3/c-erbB-3, Xmrk, an insulin receptor, an IGF-1 receptor, IRR, PDGFreceptor, a CSF-1 receptor, c-kit, STK-1/flk-2, an FGF receptor, flg,bek, an NGF receptor, Ror1 and Ror2.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising E6 or E7 genes of human papillomavirus.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising a tumor antigen.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising glucocerebrosidase.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising asparaginase.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a receiver comprising arginine deiminase.

Provided herein are compositions containing functional erythroid cellscomprising a receiver having functional activities that are either i)not present in native erythroid cells of the same lineage, or ii)present in native erythroid cells of the same lineage in reduced levelsor reduced activity levels as compared to the erythroid cells comprisingthe receiver. Such functional activities include complement inhibition,immune complex clearance, artificial antigen presentation, modulation ofthe coagulation cascade, oxygen transfer, drug delivery, cytotoxinadsorption, avoidance of phagocytosis, and extension of circulationtime.

In some embodiments, functional erythroid cells have higher levels of acomplement receptor polypeptide, such as CR1, than native erythroidcells of the same lineage by virtue of comprising a CR-1 receiver. In analternative embodiment, the functional erythroid cells comprising areceiver have higher levels of a complement receptor agonist polypeptideor complement associated polypeptide than native erythroid cells of thesame lineage, including but not limited to, the polypeptides listed intable 7 and table 10. The complement receptor receiver polypeptidecomprises a human Complement Receptor-1 (CR1) polypeptide, variant, orfunctional fragment thereof. The CR1 receiver polypeptide may be derivedfrom one or more than one of the native alleles of CR1, e.g., the Aallele (also termed the F allele or CR1*1 allele), the B allele (alsotermed the S allele or CR1*2 allele), the C allele (also termed the F′allele or CR1*3 allele), or the D allele (also termed the CR1*4 allele).The sequences and database accession numbers for these native forms areprovided in table 4. In some embodiments, the CR1 receiver polypeptidecontains a domain of a CR1 polypeptide. For example, the CR1 polypeptidemay comprise one or more short consensus repeat (SCR) domains, alsotermed complement control protein (CCP) modules or Sushi domains, e.g.,Genbank accession number AAV65577.1. In one embodiment, the CR1 receiverpolypeptide comprises one or more Short Consensus Repeats (SCRs), e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44 or greater than 44 SCRs. In another embodiment,the CR1 receiver polypeptide comprises one or more long homologousrepeat (LHR) units of CR1, e.g., LHR-A, LHR-B, LHR-C, or LHR-D, e.g., 1,2, 3, 4, 5, 6 or greater than 6 LHR domains. In another embodiment, theCR1 receiver polypeptide may comprise one or more than one extracellulardomains of CR1 fused to another cell membrane protein, e.g., glycophorinA, glycophorin B, glycophorin C, glycophorin D, kell, band 3, aquaporin1, glut 1, kidd antigen protein, rhesus antigen, including, but notlimited to the cell surface moieties listed in table 1 and table 7.

In some embodiments, a functional erythroid cell contains an exogenousnucleic acid encoding a complement receptor receiver polypeptide, oralternatively or in combination, a complement receptor agonist receiverpolypeptide or complement associated receiver polypeptide including butnot limited to, the polypeptides, and agonists to the polypeptides,listed in table 10. In some embodiments, the functional erythroid cellsfurther contain an exogenous decay-accelerating factor (CD59, GenBank:CAG46523.1) polypeptide, or an exogenous membrane cofactor (CD46,GenBank: BAA12224.1) polypeptide, or a variant or functional fragmentthereof, or a combination thereof.

CR1 activities include binding to C3b-containing immune complexes andshuttling of these immune complexes from circulation to liver and spleenmacrophages of the reticuloendothelial system. Upon encounter with cellsof the reticuloendothelial system, the immune complex is endocytosed bythe phyagocytic cell but the red blood cell is spared to continue itscirculation. The removal of the immune complex sometimes results inproteolytic cleavage of CR1 from the surface of the red blood cell. Tomeasure binding activity, one can perform an in vitro binding assaybetween erythroid cells and immune complexes. To measure sparing of theerythroid cell, one can perform an in vitro phagocytosis assay withphagocytic cells and immune complex-loaded erythroid cells. To measurein vivo clearance of circulating immune complexes to the liver, one canperform a clearance and biodistribution assay using radiolabeled immunecomplexes.

Provided are compositions containing functional erythroid cellscontaining a receiver comprising a native polypeptide at a level greaterthan that of a hematopoietic cell of the same lineage not comprising thereceiver polypeptide. For example, populations of functional erythroidcells contain receivers, such as complement receptor 1 levels at leastabout 1.1, e.g., 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 timesgreater than corresponding hematopoietic cells of the same lineage thatlack the CR1 receiver polypeptide. CR1 levels on reticulocytes anderythrocytes are typically between 50-2000 molecules per cell(Lach-Trifilieff, J Immunol 1999, 162:7549). Provided are compositionsthat contain populations of functional erythroid cells with CR1 levelsof at least about 2500, 5000, 6000, 7000, 8000, 9000, 10000, 15000,20000, 25000, 30000, 40000, 50000, 100000, 200000, 300000, 400000,500000, 600000, 700000, 800000, 900000, 1000000, or more than 1000000molecules per cell. CR1 levels in wild-type and syntheticmembrane-receiver polypeptide complexes can be measured and quantifiedby, for example, flow cytometry with antibodies specific for CR1.

In some embodiments, the receiver interacts with a circulating pathogen,such as a virus or a bacterium. In some embodiments, the functionalerythroid cell expresses an exogenous gene encoding an antibody, scFv,or nanobody specific for the circulating pathogen. The antibody, scFv,or nanobody may be expressed as a fusion protein. In other embodiments,the antibody, scFv, or nanobody receiver or another receiver withaffinity to circulating pathogens is loaded into or onto the erythroidcell. The antibody, scFv, or nanobody receiver or the other receiverwith affinity to circulating pathogens may be localized intracellularlyor extracellularly. In some embodiments, the receiver is specific for aviral or bacterial antigen, such as a surface, envelope or capsidantigen.

In some embodiments, the receiver interacts with a toxin, preferably aforeign toxin, such as derived from a pathogen or otherwise from theenvironment. In some embodiments, the functional erythroid cellexpresses a exogenous gene encoding a receiver comprising an amino acidsequence derived from lipopolysaccharide-binding protein (LBP),bactericidal/permeability-increasing protein (BPI), amyloid P component,or a cationic protein. Toxin-binding receivers may be expressed as afusion protein. In other embodiments, toxin-binding receivers may beloaded into or onto the erythroid cell. Toxin-binding receivers may belocalized intracellularly or extracellularly. In some embodiments, thetoxin binding receiver is specific for a bacterial toxin such asbotulinum or anthrax.

Further, synthetic membrane-receiver complexes may express a receivercapable of enhancing its ability to sequester a target. Potentialsequestration enhancement receivers include the polypeptide transportersincluding, but not limited to, those in table 1.

In one embodiment, the receiver comprises a polypeptide that comprisesan amino acid sequence derived from Duffy Antigen Receptor forChemokines (DARC). In one embodiment, the functional erythroid cellexpresses a exogenous gene encoding an amino acid sequence derived fromDuffy Antigen Receptor for Chemokines (DARC). The DARC receiver may beexpressed as a full-length protein or a fragment thereof. DARC may beexpressed as a fusion protein. In other embodiments, DARC protein isloaded into or onto the erythroid cell. In some embodiments, the loadedDARC is additionally functionalized or otherwise modified. The DARCreceiver molecule may be localized intracellularly or extracellularly.

DARC was identified as a potent multi-ligand chemokine receptor. DARCbelongs to the family of rhodopsin-like seven-helix transmembraneproteins. Besides erythrocytes DARC is expressed in post capillaryvenular endothelial cells, which are the primary site of leukocytetransmigration in most tissues. DARC provides a highly specific bindingsite for both CC and CXC chemokines. DARC is thought to possess a higheraffinity for ELR motif CXC chemokines. CXC chemokines are neutrophilchemoattractants and may potentially be pro-angiogenic.

Interaction between DARC and CXCL8 has demonstrated a dissociationconstant (Kd) of 5 nmol/L and receptor binding sites estimated at1000-9000 per erythrocyte (Hadley, Blood, 1997) Unlike otherseven-transmembrane chemokine receptors, DARC lacks the highly conservedG protein coupling motif located in the second cytoplasmic loop (Meny,Immunohematology, 2010). DARC is not G-protein coupled and has no knownalternative signaling mechanism. The biological role of DARC is notfully understood. DARC is thought to be a) multi-specific; b) unable toinitiate intracellular signals, and c) chemokines bound to erythrocytesurface are believed to be inaccessible to their normal targetinflammatory cells (Neote, J Biol Chem, 1993). Erythrocytes may play arole in the regulation of inflammatory processes through the presence ofDARC

Inflammatory signaling molecules, such as cytokines, can trigger localand systemic tissue damage when present in high concentrations. Burstsof cytokines are implicated in the pathogenesis of bacterial sepsis,rheumatoid arthritis, and several other inflammatory diseases.Functional erythroid cells that exogenously express natural cytokinereceptors or synthetic antibody-like receptor mimics can sequester theinflammatory cytokines. An exemplary chemokine receptor is DARC.Provided herein are functional erythroid cells comprising a receiverthat is a cytokine receptor or chemokine receptor, including, but notlimited to DARC. For example, functional erythroid cells expressing DARCreceiver (thereby increasing the amount present on native erythrocytes)may be used to modulate chemokine levels in circulation and/or withinthe body's peripheral tissues. The functional erythroid cells comprisinga DARC receiver can either be marked for destruction or can slowlyrelease the inflammatory mediators back into circulation, but at a lowand diffuse concentration. The functional erythroid cell comprising areceiver that comprises a chemokine or cytokine receptor may act as areservoir for signal transduction peptides.

In one embodiment, the receiver comprises a polypeptide that comprisesan amino acid sequence derived from an antibody. In one embodiment, thefunctional erythroid cell expresses a exogenous gene encoding an aminoacid sequence derived from an antibody. The antibody receiver may beexpressed as a full-length protein or a fragment thereof. The antibodymay be expressed as a fusion protein. In other embodiments, the antibodyprotein is loaded into or onto the erythroid cell. In some embodiments,the loaded antibody is additionally functionalized or otherwisemodified. The antibody receiver may be localized intracellularly orextracellularly. In one embodiment, the receiver comprises an antibodyamino acid sequence that is specific for a desired target. In someembodiments, the antibody is a scFv. In other embodiments, the antibodyis a nanobody.

In certain embodiments, the functional erythroid cells comprise areceiver that comprises an antibody or fragment thereof that is specificfor a target and is located on the cell surface. For example, a variablefragment (Fv) of an antibody specific for botulinum toxin binding isexpressed on the surface of the erythroid cell. Botulinum toxin bindingantibodies are known in the art (Amersdorfer, Inf and Immunity, 1997),as is the expression of the Fv portion of an antibody (Hoedemaeker,Journ of Bio Chemistry, 1997). Upon binding, the toxin is retained bythe erythroid cell through the Fv region, sequestered and shuttled viathe circulatory system to the liver for clearance from the body.

In one embodiment, the receiver comprises a polypeptide that comprisesan amino acid sequence derived from a scFv antibody. In one embodiment,the functional erythroid cell expresses a exogenous gene encoding anamino acid sequence derived from a scFv antibody. The scFv antibodyreceiver may be expressed as a full-length protein or a fragmentthereof. The scFv antibody may be expressed as a fusion protein. Inother embodiments, the scFv protein is loaded into or onto the erythroidcell. Suitable scFv receiver polypeptides that may be expressed byfunctional erythroid cells include, but are not limited to, those listedin table 7.

scFv antibodies have been constructed mainly from hybridoma, spleencells from immunized mice, and B lymphocytes from human. The variableregion of an antibody is formed by the noncovalent heterodimer of thevariable domains of the V(H) and V(L) domains, which can then be used inthe construction of a recombinant scFv antibody.

The production of scFvs is known in the art and require mRNA to first beisolated from hybridoma (or also from the spleen, lymph cells, and bonemorrow) followed by reverse transcription into cDNA to serve as atemplate for antibody gene amplification (PCR). With this method, largelibraries with a diverse set of antibody-derived scFvs (a set comparableto that of the original antibodies from which the scFvs are modeled) canbe created.

The scFv receiver may be made specific to any target molecule including,but not limited to, those in table 5.

In one example, a scFv receiver specific for anthrax toxin may beexpressed on a functional erythroid cell. Upon administration to asubject in need thereof an effective dose of a population of erythroidcell comprising a receiver molecule specific for anthrax toxin can beused to capture and sequester the anthrax toxin. The erythroid cellmigrates to the liver where clearance occurs.

In certain embodiments, erythrocytes comprise a receiver comprising acamelid-derived nanobody expressed on the surface of the cell.Nanobodies are usually 12-15 kDa. They are considerably smaller thanantibodies and scFv. Nanobodies may thus be easier to transfect, and thenanobody receiver will be more easily expressed, translated and ortransported to the cell surface in an erythroid cell. In certainembodiments, nanobody receivers are employed to minimize immunogeniceffects caused by a specific receiver. Nanobodies because of their smallsize will offer reduced immunogenic potential. In certain embodiments,receiver nanobodies are employed because they limit changes in themechanical and morphological behavior of the plasma membrane of thefunctional erythroid cell. This may allow the functional erythroid cellto exhibit normal circulatory red blood cell behavior. In certainembodiments, receiver nanobodies are employed because they have anincreased ability to recognize hidden or uncommon epitopes compared tostandard antibodies. For example, they can bind to small enzymaticcavities of a target and modulate the molecular behavior of the target.

In certain embodiments, functional erythroid cells comprise receivernanobodies with specificity to target epitopes of molecules in the humancomplement system. Such functional erythroid cells may be administeredto a subject in need thereof to selectively deplete one or moreover-active factors of the complement system. For example, C5 may betargeted by erythroid cells comprising receiver nanobodies withspecificity to target epitopes of C5 and cleared from the system by theerythroid cells upon administration of the cells into a subject. Thisapproach is suitable to provide a therapeutic effect, e.g., for acomplement disorder, such as paroxysmal nocturnal hemoglobinuria. Incertain embodiments, functional erythroid cells comprise receivernanobodies with specificity to target epitopes of molecules including,but not limited to, those listed in table 5.

In some embodiments, the receiver comprises a polypeptide that comprisesan amino acid sequence derived from one of proteases, nucleases,amylase, lyase (sucrase) or hydrolase (DNase, lipase). In oneembodiment, the functional erythroid cell expresses a exogenous geneencoding an amino acid sequence derived from one of proteases,nucleases, amylase, lyase (sucrase) or hydrolase (DNase, lipase).Receiver proteases, nucleases, amylases, lyases and hydrolases may beexpressed as a full-length protein or a fragment thereof. Receiverproteases, nucleases, amylases, lyases and hydrolases may be expressedas a fusion protein. In other embodiments, receiver proteases,nucleases, amylases, lyases or hydrolases are loaded into or onto theerythroid cell. In some embodiments, the loaded receiver proteases,nucleases, amylases, lyases or hydrolases are additionallyfunctionalized or otherwise modified. The receiver protease, nuclease,amylase, lyase or hydrolase receiver molecule may be localizedintracellularly or extracellularly.

In certain embodiments, functional erythroid cells comprise a receivercomprising a protease, a nuclease, an amylase, a lyase or a hydrolase.The functional erythroid cell comprising a protease, a nuclease, anamylase, a lyase or a hydrolase receiver is capable of degrading atarget on the erythroid cell independent of circulatory clearance, e.g.,by macrophages in the liver. In certain embodiments, functionalerythroid cells comprising a receiver comprising a protease, a nuclease,an amylase, a lyase or a hydrolase may be administered to a subject inneed thereof to treat a cancer by selectively degrading metabolites thatare essential for cancer cell growth. For example, asparaginase is usedto decrease local asparagine levels to treat acute lymphoblasticleukemia and acute myeloid leukemia. Suitable receivers may, e.g.,comprise one or both of the two major classes of enzymes capable ofdegrading target molecules, lyases and hydrolases. In certainembodiments, functional erythroid cells are provided comprising areceiver comprising a molecule including but not limited to those listedin table 7.

In certain embodiments, erythrocytes comprise a receiver comprising alyase. In one embodiment, the lyase is valine decarboxylase. Valinedecarboxylase receiver may be expressed within the intracellular spaceof the erythroid cell. Functional erythroid cells comprising a valinedecarboxylase receiver may be administered to a subject in need thereofto modulate valine levels within the blood. Erythroid cells comprising avaline decarboxylase receiver are suitable to treat valinemia, aninherited disorder that increases levels of the amino acid valine in theblood. Affected individuals typically develop vomiting, failure tothrive, intellectual disability, and fatigue. Valinemia is caused by adeficiency of the valine transaminase enzyme and has an autosomalrecessive pattern of inheritance.

In certain embodiments, erythrocytes comprise a receiver comprising ahydrolase. In one embodiment, the hydrolase is deoxyribonuclease I(DNase I). DNase I receiver may be expressed on the surface of theerythroid cell. Functional erythroid cells comprising a DNase I receivermay be administered to a subject in need thereof to preferentiallycleave circulating DNA at phosphodiester linkages adjacent to apyrimidine nucleotide, yielding 5′-phosphate-terminated polynucleotideswith a free hydroxyl group on position 3′. On average tetra-nucleotidesare produced. Erythroid cells comprising a DNase I receiver are suitableto treat conditions exacerbated by high levels of immunogenic DNA incirculation, such as systemic lupus erythematosus (SLE).

In certain embodiments the receiver is capable of responding to anexternal stimulus, e.g., upon binding to a ligand or contacting thestimulus, wherein responding entails, for example, moving, re-folding,changing conformation, forming a dimer, forming a homodimer, forming aheterodimer, forming a multimer, transducing a signal, emitting energyin a detectable form (e.g., fluorescence), functionally interacting withanother receiver, or functionally interacting with a non-receiverpolypeptide.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a fusion molecule capable of promoting fusion of the syntheticmembrane-receiver complex to a target cell that is i) different fromand/or ii) acts independent of the receiver, wherein the receiver iscapable of interacting with a target. In some embodiments, the syntheticmembrane-receiver complex does not comprise a receiver comprisingSyncytin-1.

In some embodiments, the synthetic membrane-receiver complex does notcomprise a photosensitive synthetic compound, such as, e.g. a compoundthat can be activated by photons or quenchable compounds. In someembodiments, the synthetic membrane-receiver complex does not comprisean activatable molecular detection agent capable of producing adetectable response. In some embodiments, the syntheticmembrane-receiver complex does not comprise a diagnostic compound. Insome embodiments, the synthetic membrane-receiver complex does notcomprise a virus or bacterium.

Receiver Contacting

In certain embodiments, the polypeptide receiver is expressed within thesynthetic membrane-receiver polypeptide complex. The polypeptidereceiver may be exhibited on the surface of the syntheticmembrane-receiver polypeptide complex or may reside within the syntheticmembrane-receiver polypeptide complex.

In certain embodiments, the polypeptide receiver is conjugated to thesynthetic membrane-receiver polypeptide complex. The polypeptidereceiver usually is conjugated to the surface of the syntheticmembrane-receiver polypeptide complex. Conjugation may be achievedchemically or enzymatically, by methods known in the art and describedherein. Non-polypeptide receivers may also be conjugated to a syntheticmembrane-receiver complex. In some embodiments, the receiver is notconjugated to the synthetic membrane-receiver complex.

In certain embodiments, the polypeptide receiver is loaded into thesynthetic membrane-receiver polypeptide complex. Non-polypeptidereceivers may also be loaded within a synthetic membrane-receivercomplex. In some embodiments, the receiver is not loaded into or ontothe synthetic membrane-receiver complex.

In some embodiments, the synthetic membrane-receiver complex comprises areceiver that is optionally expressed from an exogenous nucleic acid,conjugated to the complex, loaded into or onto the complex, and anycombination thereof. Optionally, the synthetic membrane-receiver complexcomprises a therapeutic agent or other payload.

In some embodiments, the synthetic membrane-receiver complex isgenerated by contacting a suitable isolated cell, e.g., an erythroidcell, a reticulocyte, an erythroid cell precursor, a platelet, or aplatelet precursor, with an exogenous nucleic acid encoding a receiverpolypeptide. In some embodiments, the receiver polypeptide is encoded bya DNA, which is contacted with a nucleated erythroid precursor cell or anucleated platelet precursor cell. In some embodiments, the receiverpolypeptide is encoded by an RNA, which is contacted with a platelet, anucleate erythroid cell, a nucleated platelet precursor cell, or areticulocyte. In some embodiments, the receiver is a polypeptide, whichis contacted with a primary platelet, a nucleated erythroid cell, anucleated platelet precursor cell, a reticulocyte, or an erythrocyte.

A receiver polypeptide may be expressed from a transgene introduced intoan erythroid cell by electroporation, chemical or polymerictransfection, viral transduction, mechanical membrane disruption, orother method; a receiver polypeptide that is expressed from mRNA that isintroduced into a cell by electroporation, chemical or polymerictransfection, viral transduction, mechanical membrane disruption, orother method; a receiver polypeptide that is over-expressed from thenative locus by the introduction of an external factor, e.g., atranscriptional activator, transcriptional repressor, or secretorypathway enhancer; and/or a receiver polypeptide that is synthesized,extracted, or produced from a production cell or other external systemand incorporated into the erythroid cell.

In certain embodiments, the polypeptide receiver is expressed within thesynthetic membrane-receiver polypeptide complex. The polypeptidereceiver may be exhibited on the surface of the syntheticmembrane-receiver polypeptide complex or may reside within the syntheticmembrane-receiver polypeptide complex.

In certain embodiments, the synthetic membrane-receiver polypeptidecomplex is a cell, e.g., an erythroid cell or a platelet expressing areceiver polypeptide. Receiver polypeptides can be introduced bytransfection of single or multiple copies of genes, transduction with avirus, or electroporation in the presence of DNA or RNA. Methods forexpression of exogenous proteins in mammalian cells are well known inthe art. For example, expression of exogenous factor IX in hematopoieticcells is induced by viral transduction of CD34+ progenitor cells, seeChang et al., Nat Biotechnol 2006, 24:1017.

Nucleic acids such as DNA expression vectors or mRNA for producing thereceiver polypeptide may be introduced into progenitor cells (e.g., anerythroid cell progenitor or a platelet progenitor and the like) thatare suitable to produce the synthetic membrane-receiver polypeptidecomplexes described herein. The progenitor cells can be isolated from anoriginal source or obtained from expanded progenitor cell population viaroutine recombinant technology as provided herein. In some instances,the expression vectors can be designed such that they can incorporateinto the genome of cells by homologous or non-homologous recombinationby methods known in the art.

In some instances, e.g., for a synthetic membrane-receiver polypeptidecomplex that is an erythroid cell comprising a receiver polypeptide, anucleic acid encoding a non-receiver polypeptide that can selectivelytarget and cut the genome, for example a CRISPR/Cas9, transcriptionalactivator-like effector nuclease (TALEN), or zinc finger nuclease, isused to direct the insertion of the exogenous nucleic acid of theexpression vector encoding the receiver polypeptide to a particulargenomic location, for example the CR1 locus (1q32.2), the hemoglobinlocus (11p15.4), or another erythroid-associated protein including, butnot limited to, those listed in table 1 and table 3.

In some instances, the exogenous nucleic acid is an RNA molecule, or aDNA molecule that encodes for an RNA molecule, that silences orrepresses the expression of a target gene. For example, the molecule canbe a small interfering RNA (siRNA), an antisense RNA molecule, or ashort hairpin RNA (shRNA) molecule.

Methods for transferring expression vectors into progenitor cells thatare suitable to produce the synthetic membrane-receiver polypeptidecomplexes described herein include, but are not limited to, viralmediated gene transfer, liposome mediated transfer, transformation, geneguns, transfection and transduction, e.g., viral mediated gene transfersuch as the use of vectors based on DNA viruses such as adenovirus,adenoassociated virus and herpes virus, as well as retroviral basedvectors. Examples of modes of gene transfer include e.g., naked DNA,CaPO₄ precipitation, DEAE dextran, electroporation, protoplast fusion,lipofection, and cell microinjection.

A progenitor cell subject to transfer of an exogenous nucleic acid thatencodes a polypeptide receiver can be cultured under suitable conditionsallowing for differentiation into mature enucleated red blood cells,e.g., the in vitro culturing process described herein. The resultingenucleated red blood cells display proteins associated with matureerythrocytes, e.g., hemoglobin, glycophorin A, and receiver polypeptideswhich can be validated and quantified by standard methods (e.g., Westernblotting or FACS analysis). Isolated mature enucleated red blood cellscomprising a receiver and platelets comprising a receiver are twoexamples of synthetic membrane-receiver polypeptide complexes of theinvention.

In some embodiments, the synthetic membrane-receiver complex isgenerated by contacting a reticulocyte with an exogenous nucleic acidencoding a receiver polypeptide. In some embodiments, the receiverpolypeptide is encoded by an RNA which is contacted with a reticulocyte.In some embodiments, the receiver is a polypeptide which is contactedwith a reticulocyte.

Isolated reticulocytes may be transfected with mRNA encoding a receiverpolypeptide to generate a synthetic membrane-receiver complex. MessengerRNA may be derived from in vitro transcription of a cDNA plasmidconstruct containing the coding sequence corresponding to the receiverpolypeptide. For example, the cDNA sequence corresponding to thereceiver polypeptide may be inserted into a cloning vector containing apromoter sequence compatible with specific RNA polymerases. For example,the cloning vector ZAP Express® pBK-CMV (Stratagene, La Jolla, Calif.,USA) contains T3 and T7 promoter sequence compatible with T3 and T7 RNApolymerase, respectively. For in vitro transcription of sense mRNA, theplasmid is linearized at a restriction site downstream of the stopcodon(s) corresponding to the end of the coding sequence of the receiverpolypeptide. The mRNA is transcribed from the linear DNA template usinga commercially available kit such as, for example, the RNAMaxx® HighYield Transcription Kit (from Stratagene, La Jolla, Calif., USA). Insome instances, it may be desirable to generate 5′-m7GpppG-capped mRNA.As such, transcription of a linearized cDNA template may be carried outusing, for example, the mMESSAGE mMACHINE High Yield Capped RNATranscription Kit from Ambion (Austin, Tex., USA). Transcription may becarried out in a reaction volume of 20-100 μl at 37° C. for 30 min to 4h. The transcribed mRNA is purified from the reaction mix by a brieftreatment with DNase I to eliminate the linearized DNA template followedby precipitation in 70% ethanol in the presence of lithium chloride,sodium acetate or ammonium acetate. The integrity of the transcribedmRNA may be assessed using electrophoresis with an agarose-formaldehydegel or commercially available Novex pre-cast TBE gels (e.g., Novex,Invitrogen, Carlsbad, Calif., USA).

Messenger RNA encoding the receiver polypeptide may be introduced intoreticulocytes using a variety of approaches including, for example,lipofection and electroporation (van Tandeloo et al., Blood 98:49-56(2001)). For lipofection, for example, 5 μg of in vitro transcribed mRNAin Opti-MEM (Invitrogen, Carlsbad, Calif., USA) is incubated for 5-15min at a 1:4 ratio with the cationic lipid DMRIE-C(Invitrogen).Alternatively, a variety of other cationic lipids or cationic polymersmay be used to transfect cells with mRNA including, for example, DOTAP,various forms of polyethylenimine, and polyL-lysine (Sigma-Aldrich,Saint Louis, Mo., USA), and Superfect (Qiagen, Inc., Valencia, Calif.,USA; See, e.g., Bettinger et al., Nucleic Acids Res. 29:3882-3891(2001)). The resulting mRNA/lipid complexes are incubated with cells(1-2×10⁶ cells/ml) for 2 h at 37° C., washed and returned to culture.For electroporation, for example, about 5 to 20×10⁶ cells in 500 μl ofOpti-MEM (Invitrogen, Carlsbad, Calif., USA) are mixed with about 20 μgof in vitro transcribed mRNA and electroporated in a 0.4-cm cuvetteusing, for example, and Easyject Plus device (EquiBio, Kent, UnitedKingdom). In some instances, it may be necessary to test variousvoltages, capacitances and electroporation volumes to determine theuseful conditions for transfection of a particular mRNA into areticulocyte. In general, the electroporation parameters required toefficiently transfect cells with mRNA appear to be less detrimental tocells than those required for electroporation of DNA (van Tandeloo etal., Blood 98:49-56 (2001)).

Alternatively, mRNA may be transfected into a reticulocyte using apeptide-mediated RNA delivery strategy (See, e.g., Bettinger et al.,Nucleic Acids Res. 29:3882-3891 (2001)). For example, the cationic lipidpolyethylenimine 2 kDA (Sigma-Aldrich, Saint Louis, Mo., USA) may becombined with the melittin peptide (Alta Biosciences, Birmingham, UK) toincrease the efficiency of mRNA transfection, particularly inpost-mitotic primary cells. The mellitin peptide may be conjugated tothe PEI using a disulfide cross-linker such as, for example, thehetero-bifunctional cross-linker succinimidyl 3-(2-pyridyldithio)propionate. In vitro transcribed mRNA is preincubated for 5 to 15 minwith the mellitin-PEI to form an RNA/peptide/lipid complex. This complexis then added to cells in serum-free culture medium for 2 to 4 h at 37°C. in a 5% CO₂ humidified environment and then removed and thetransfected cells allowed to continue growing in culture.

In some embodiments, the synthetic membrane-receiver complex isgenerated by contacting a suitable isolated erythroid cell precursor ora platelet precursor with an exogenous nucleic acid encoding a receiverpolypeptide. In some embodiments, the receiver polypeptide is encoded bya DNA, which is contacted with a nucleated erythroid precursor cell or anucleated platelet precursor cell. In some embodiments, the receiverpolypeptide is encoded by an RNA, which is contacted with a platelet, anucleate erythroid cell, or a nucleated platelet precursor cell.

Receivers may be genetically introduced into erythroid cell precursors,platelet precursor, or nucleated erythroid cells prior to terminaldifferentiation using a variety of DNA techniques, including transientor stable transfections and gene therapy approaches. The receiverpolypeptides may be expressed on the surface and/or in the cytoplasm ofmature red blood cell or platelet.

Viral gene transfer may be used to transfect the cells with DNA encodinga receiver polypeptide. A number of viruses may be used as gene transfervehicles including Moloney murine leukemia virus (MMLV), adenovirus,adeno-associated virus (AAV), herpes simplex virus (HSV), lentivirusessuch as human immunodeficiency virus 1 (HIV 1), and spumaviruses such asfoamy viruses, for example (See, e.g., Osten et al., HEP 178:177-202(2007)). Retroviruses, for example, efficiently transduce mammaliancells including human cells and integrate into chromosomes, conferringstable gene transfer.

A receiver polypeptide may be transfected into an erythroid cellprecursor, a platelet precursor, or a nucleated erythroid cell,expressed and subsequently retained and exhibited in a mature red bloodcell or platelet. A suitable vector is the Moloney murine leukemia virus(MMLV) vector backbone (Malik et al., Blood 91:2664-2671 (1998)).Vectors based on MMLV, an oncogenic retrovirus, are currently used ingene therapy clinical trials (Hossle et al., News Physiol. Sci. 17:87-92(2002)). For example, a DNA construct containing the cDNA encoding areceiver polypeptide can be generated in the MMLV vector backbone usingstandard molecular biology techniques. The construct is transfected intoa packaging cell line such as, for example, PA317 cells and the viralsupernatant is used to transfect producer cells such as, for example,PG13 cells. The PG13 viral supernatant is incubated with an erythroidcell precursor, a platelet precursor, or a nucleated erythroid cell thathas been isolated and cultured or has been freshly isolated as describedherein. The expression of the receiver polypeptide may be monitoredusing FACS analysis (fluorescence-activated cell sorting), for example,with a fluorescently labeled antibody directed against the receiverpolypeptide, if it is located on the surface of the syntheticmembrane-receiver polypeptide complex. Similar methods may be used toexpress a receiver polypeptide that is located in the inside of thesynthetic membrane-receiver polypeptide complex.

Optionally, a fluorescent tracking molecule such as, for example, greenfluorescent protein (GFP) may be transfected using a viral-basedapproach (Tao et al., Stem Cells 25:670-678 (2007)). Ecotopic retroviralvectors containing DNA encoding the enhanced green fluorescent protein(EGFP) or a red fluorescent protein (e.g., DsRed-Express) are packagedusing a packaging cell such as, for example, the Phoenix-Eco cell line(distributed by Orbigen, San Diego, Calif.). Packaging cell lines stablyexpress viral proteins needed for proper viral packaging including, forexample, gag, pol, and env. Supernatants from the Phoenix-Eco cells intowhich viral particles have been shed are used to transduce e.g.,erythroid cell precursors, platelet precursors, or a nucleated erythroidcells. In some instances, transduction may be performed on a speciallycoated surface such as, for example, fragments of recombinantfibronectin to improve the efficiency of retroviral mediated genetransfer (e.g., RetroNectin, Takara Bio USA, Madison, Wis.). Cells areincubated in RetroNectin-coated plates with retroviral Phoenix-Ecosupernatants plus suitable co-factors. Transduction may be repeated thenext day. In this instance, the percentage of cells expressing EGFP orDsRed-Express may be assessed by FACS. Other reporter genes that may beused to assess transduction efficiency include, for example,beta-galactosidase, chloramphenicol acetyltransferase, and luciferase aswell as low-affinity nerve growth factor receptor (LNGFR), and the humancell surface CD24 antigen (Bierhuizen et al., Leukemia 13:605-613(1999)).

Nonviral vectors may be used to introduce genetic material into suitableerythroid cells, platelets or precursors thereof to generate syntheticmembrane-receiver polypeptide complexes. Nonviral-mediated gene transferdiffers from viral-mediated gene transfer in that the plasmid vectorscontain no proteins, are less toxic and easier to scale up, and have nohost cell preferences. The “naked DNA” of plasmid vectors is by itselfinefficient in delivering genetic material encoding a receiverpolypeptide to a cell and therefore is combined with a gene deliverymethod that enables entry into cells. A number of delivery methods maybe used to transfer nonviral vectors into suitable erythroid cells,platelets or precursors thereof including chemical and physical methods.

A nonviral vector encoding a receiver polypeptide may be introduced intosuitable erythroid cells, platelets or precursors thereof usingsynthetic macromolecules such as cationic lipids and polymers(Papapetrou et al., Gene Therapy 12:S118-S130 (2005)). Cationicliposomes, for example form complexes with DNA through chargeinteractions. The positively charged DNA/lipid complexes bind to thenegative cell surface and are taken up by the cell by endocytosis. Thisapproach may be used, for example, to transfect hematopoietic cells(See, e.g., Keller et al., Gene Therapy 6:931-938 (1999)). For erythroidcells, platelets or precursors thereof the plasmid DNA (approximately0.5 μg in 25-100 μL of a serum free medium, such as, for example,OptiMEM (Invitrogen, Carlsbad, Calif.)) is mixed with a cationicliposome (approximately 4 μg in 25 μL of serum free medium) such as thecommercially available transfection reagent Lipofectamine™ (Invitrogen,Carlsbad, Calif.) and allowed to incubate for at least 20 min to formcomplexes. The DNA/liposome complex is added to suitable erythroidcells, platelets or precursors thereof and allowed to incubate for 5-24h, after which time transgene expression or the receiver polypeptide maybe assayed. Alternatively, other commercially available liposometranfection agents may be used (e.g., In vivo GeneSHUTTLE™, Qbiogene,Carlsbad, Calif.).

Optionally, a cationic polymer such as, for example, polyethylenimine(PEI) may be used to efficiently transfect erythroid cell progenitorcells, for example hematopoietic and umbilical cord blood-derived CD34+cells (See, e.g., Shin et al., Biochim Biophys. Acta 1725:377-384(2005)). Human CD34+ cells are isolated from human umbilical cord bloodand cultured in Iscove's modified Dulbecco's medium supplemented with200 ng/ml stem cell factor and 20% heat-inactivated fetal bovine serum.Plasmid DNA encoding the receiver polypeptide is incubated with branchedor linear PEIs varying in size from 0.8 K to 750 K (Sigma Aldrich, SaintLouis, Mo., USA; Fermetas, Hanover, Md., USA). PEI is prepared as astock solution at 4.2 mg/ml distilled water and slightly acidified to pH5.0 using HCl. The DNA may be combined with the PEI for 30 min at roomtemperature at various nitrogen/phosphate ratios based on thecalculation that 1 μg of DNA contains 3 nmol phosphate and 1 μl of PEIstock solution contains 10 nmol amine nitrogen. The isolated CD34+ cellsare seeded with the DNA/cationic complex, centrifuged at 280×g for 5 minand incubated in culture medium for 4 or more h until gene expression ofthe receiver polypeptide is assessed.

A plasmid vector may be introduced into suitable erythroid cells,platelets or precursors thereof using a physical method such asparticle-mediated transfection, “gene gun”, biolistics, or particlebombardment technology (Papapetrou, et al., (2005) Gene Therapy12:S118-S130). In this instance, DNA encoding the receiver polypeptideis absorbed onto gold particles and administered to cells by a particlegun. This approach may be used, for example, to transfect erythroidprogenitor cells, e.g., hematopoietic stem cells derived from umbilicalcord blood (See, e.g., Verma et al., Gene Therapy 5:692-699 (1998)). Assuch, umbilical cord blood is isolated and diluted three fold inphosphate buffered saline. CD34+ cells are purified using an anti-CD34monoclonal antibody in combination with magnetic microbeads coated witha secondary antibody and a magnetic isolation system (e.g., MiltenyiMiniMac System, Auburn, Calif., USA). The CD34+ enriched cells may becultured as described herein. For transfection, plasmid DNA encoding thereceiver polypeptide is precipitated onto a particle, for example goldbeads, by treatment with calcium chloride and spermidine. Followingwashing of the DNA-coated beads with ethanol, the beads may be deliveredinto the cultured cells using, for example, a Biolistic PDS-1000/HeSystem (Bio-Rad, Hercules, Calif., USA). A reporter gene such as, forexample, beta-galactosidase, chloramphenicol acetyltransferase,luciferase, or green fluorescent protein may be used to assessefficiency of transfection.

Optionally, electroporation methods may be used to introduce a plasmidvector into suitable erythroid cells, platelets or precursors thereof.Electroporation creates transient pores in the cell membrane, allowingfor the introduction of various molecules into the cells including, forexample, DNA and RNA as well as antibodies and drugs. As such, CD34+cells are isolated and cultured as described herein Immediately prior toelectroporation, the cells are isolated by centrifugation for 10 min at250×g at room temperature and resuspended at 0.2-10×10̂6 viable cells/mlin an electroporation buffer such as, for example, X-VIVO 10supplemented with 1.0% human serum albumin (HSA). The plasmid DNA (1-50μg) is added to an appropriate electroporation cuvette along with 500 μlof cell suspension. Electroporation may be done using, for example, anECM 600 electroporator (Genetronics, San Diego, Calif., USA) withvoltages ranging from 200 V to 280 V and pulse lengths ranging from 25to 70 milliseconds. A number of alternative electroporation instrumentsare commercially available and may be used for this purpose (e.g., GenePulser Xcell™, BioRad, Hercules, Calif.; Cellject Duo, Thermo Science,Milford, Mass.). Alternatively, efficient electroporation of isolatedCD34+ cells may be performed using the following parameters: 4 mmcuvette, 1600 μF, 550 V/cm, and 10 μg of DNA per 500 μl of cells at1×105 cells/ml (Oldak et al., Acta Biochimica Polonica 49:625-632(2002)).

Nucleofection, a form of electroporation, may also be used to transfectsuitable erythroid cells, platelets or precursors thereof. In thisinstance, transfection is performed using electrical parameters incell-type specific solutions that enable DNA (or other reagents) to bedirectly transported to the nucleus thus reducing the risk of possibledegradation in the cytoplasm. For example, a Human CD34 CellNucleofector™ Kit (from amaxa inc.) may be used to transfect suitableerythroid cells, platelets or precursors thereof. In this instance,1-5×10⁶ cells in Human CD34 Cell Nucleofector™ Solution are mixed with1-5 μg of DNA and transfected in the Nucleofector™ instrument usingpreprogrammed settings as determined by the manufacturer.

Erythroid cells, platelets or precursors thereof may be non-virallytransfected with a conventional expression vector which is unable toself-replicate in mammalian cells unless it is integrated in the genome.Alternatively, erythroid cells, platelets or precursors thereof may betransfected with an episomal vector which may persist in the hostnucleus as autonomously replicating genetic units without integrationinto chromosomes (Papapetrou et al., Gene Therapy 12:S118-S130 (2005)).These vectors exploit genetic elements derived from viruses that arenormally extrachromosomally replicating in cells upon latent infectionsuch as, for example, EBV, human polyomavirus BK, bovine papillomavirus-1 (BPV-1), herpes simplex virus-1 (HSV) and Simian virus 40(SV40). Mammalian artificial chromosomes may also be used for nonviralgene transfer (Vanderbyl et al., Exp. Hematol. 33:1470-1476 (2005)).

Exogenous nucleic acids encoding a polypeptide receiver may be assembledinto expression vectors by standard molecular biology methods known inthe art, e.g., restriction digestion, overlap-extension PCR, and Gibsonassembly.

Exogenous nucleic acids may comprise a gene encoding a polypeptidereceiver that is not normally expressed on the cell surface, e.g., of anerythroid cell, fused to a gene that encodes an endogenous or nativemembrane protein, such that the receiver polypeptide is expressed on thecell surface. For example, a exogenous gene encoding a receiverpolypeptide can be cloned at the N terminus following the leadersequence of a type 1 membrane protein, at the C terminus of a type 2membrane protein, or upstream of the GPI attachment site of a GPI-linkedmembrane protein.

Standard cloning methods can be used to introduce flexible amino acidlinkers between two fused genes. For example, the flexible linker is apoly-glycine poly-serine linker such as [Gly4Ser]3_(SEQ ID NO: 24)commonly used in generating single-chain antibody fragments fromfull-length antibodies (Antibody Engineering: Methods & Protocols, Lo2004), or ala-gly-ser-thr polypeptides such as those used to generatesingle-chain Arc repressors (Robinson & Sauer, PNAS 1998). In someembodiments, the flexible linker provides the receiver polypeptide withmore flexibility and steric freedom than the equivalent constructwithout the flexible linker. This added flexibility is useful inapplications that require binding to a target, e.g., an antibody orprotein, or an enzymatic reaction of the receiver for which the activesite must be accessible to the substrate (e.g., the target).

An epitope tag may be placed between two fused genes, such as, e.g., anucleic acid sequence encoding an HA epitope tag—amino acids YPYDVPDYA(Seq. ID No. 4), a CMyc tag—amino acids EQKLISEEDL (Seq. ID No. 5), or aFlag tag—amino acids DYKDDDDK (Seq. ID No. 6). The epitope tag may beused for the facile detection and quantification of expression usingantibodies against the epitope tag by flow cytometry, western blot, orimmunoprecipitation.

In some embodiments, the synthetic membrane-receiver polypeptidecomprises a receiver polypeptide and at least one other heterologouspolypeptide. The second polypeptide can be a fluorescent protein. Thefluorescent protein can be used as a reporter to assess transductionefficiency. In some embodiments, the fluorescent protein is used as areporter to assess expression levels of the receiver polypeptide if bothare made from the same transcript. In some embodiments, the at least oneother polypeptide is heterologous and provides a function, such as,e.g., multiple antigens, multiple capture targets, enzyme cascade. Inone embodiment, the recombinant nucleic acid comprises a gene encoding areceiver and a second gene, wherein the second gene is separated fromthe gene encoding the receiver by a viral-derived T2A sequence(gagggcagaggaagtcttctaacatgcggtgacgtggaggsgsstcccggccct (Seq. ID No. 7))that is post-translationally cleaved into two mature proteins.

In some embodiments, the receiver polypeptide is complement receptor 1(CR-1). The gene sequence for complement receptor 1 is amplified usingPCR. In some embodiments, the exogenous nucleic acid encoding a receiverpolypeptide comprises a gene sequence for a scFv against hepatitis Bantigen that is fused to the 3′ end of the sequence for Kell andamplified using PCR. In some embodiments, the exogenous nucleic acidencoding a receiver polypeptide comprises a gene sequence for a scFvagainst hepatitis B antigen that is fused to a poly-glycine/serinelinker, followed by the 3′ end of the sequence for Kell, and amplifiedusing PCR. In some embodiments, the exogenous nucleic acid encoding areceiver polypeptide comprises the 3′ end of a gene sequence for a scFvagainst hepatitis B antigen that is fused to an epitope tag sequence, ofwhich may be one, or a combination of, an; HA-tag, Green fluorescentprotein tag, Myc-tag, chitin binding protein, maltose binding protein,glutathione-S-transferase, poly(His)tag, thioredoxin, poly(NANP),FLAG-tag, V5-tag, AviTag, Calmodulin-tag, polyglutamate-tag, E-tag,S-tag, SBP-tag, Softag-1, Softag-3, Strep-tag, TC-tag, VSV-tag,Xpress-tag, Isopeptag, SpyTag, biotin carboxyl carrier protein, Nus-tag,Fc-tag, or Ty-tag. The entire construct is fused to the 3′ end of thesequence for Kell and then amplified using PCR. The exogenous geneconstructs encoding the various receiver polypeptides are, for example,subsequently loaded into a lentiviral vector and used to transduce aCD34+ cell population.

In one embodiment, the gene comprising an adenosine deaminase receiveris placed in the pSP64 vector. The vector is linearized and RNApolymerase generates mRNA coding for the receiver polypeptide. In oneembodiment, a population of neutrophils is electroporated using anIngenio electroporation kit such that 10, 100, 1,000, 10,000 TU/ml ofmRNA coding for surface expression of GluN1 receiver to generate asynthetic membrane-receiver polypeptide complex. In one embodiment, apopulation of platelet cells is incubated with Trans-IT mRNA and 10,100, or 1000 TU/ml (transducing units/ml) of mRNA coding for thymidinephosphorylase protein receiver to generate a synthetic membrane-receiverpolypeptide complex. In one embodiment, a population of erythroid cellsis incubated with lentiviral vectors comprising exogenous nucleic acidencoding a receiver polypeptide, specific plasmids of which may include;pLKO.1 puro, PLKO.1—TRC cloning vector, pSico, FUGW, pLVTHM, pLJM1,pLionll, pMD2.G, pCMV-VSV-G, pCI-VSVG, pCMV-dR8.2 dvpr, psPAX2,pRSV-Rev, and pMDLg/pRRE to generate a synthetic membrane-receiverpolypeptide complex. The vectors may be administered at 10, 100, 1,000,10,000 pfu and incubated for 12 hrs.

In one embodiment, a population of erythroid cells is incubated withLipofectamine 2000 and 10, 100, or 1000 TU/ml (transducing units/ml) ofDNA coding for oxalase receiver.

In certain embodiments, the polypeptide receiver is conjugated to thesynthetic membrane-receiver polypeptide complex. The polypeptidereceiver usually is conjugated to the surface of the syntheticmembrane-receiver polypeptide complex. Conjugation may be achievedchemically or enzymatically. Non-polypeptide receivers may also beconjugated to a synthetic membrane-receiver complex.

In some embodiments, the synthetic membrane-receiver complex comprises areceiver that is chemically conjugated. Chemical conjugation of areceiver may be accomplished by covalent bonding of the receiver toanother molecule, with or without use of a linker. The formation of suchconjugates is within the skill of artisans and various techniques areknown for accomplishing the conjugation, with the choice of theparticular technique being guided by the materials to be conjugated. Theaddition of amino acids to the polypeptide (C- or N-terminal) whichcontain ionizable side chains, e.g., aspartic acid, glutamic acid,lysine, arginine, cysteine, histidine, or tyrosine, and are notcontained in the active portion of the polypeptide sequence, serve intheir unprotonated state as a potent nucleophile to engage in variousbioconjugation reactions with reactive groups attached to polymers,e.g., homo- or hetero-bi-functional PEG (e.g., Lutolf and Hubbell,Biomacromolecules 2003; 4:713-22, Hermanson, Bioconjugate Techniques,London. Academic Press Ltd; 1996). Receiver conjugation is not notrestricted to polypeptides, e.g., a peptide ligand, an antibody, anantibody fragment, or aptamer but is applicable also for non-polypeptidereceivers, e.g., lipids, carbohydrates, nucleic acids, and smallmolecules.

In an embodiment, the receiver may be bound to the surface of asynthetic membrane-receiver complex through a biotin-streptavidinbridge. For example, a biotinylated antibody receiver may be linked to anon-specifically biotinylated surface of the synthetic membrane-receivercomplex through a streptavidin bridge. Antibodies can be conjugated tobiotin by a number of chemical means (See, e.g., Hirsch et al., MethodsMol. Biol. 295: 135-154 (2004)). Any surface membrane proteins of asynthetic membrane-receiver complex may be biotinylated using an aminereactive biotinylation reagent such as, for example, EZ-LinkSulfo-NHS-SS-Biotin (sulfosuccinimidyl2-(biotinamido)-ethyl-1,3-dithiopropionate; Pierce-Thermo Scientific,Rockford, Ill., USA; See, e.g., Jaiswal et al., Nature Biotech. 21:47-51(2003)). For example, isolated erythroid cells may be incubated for 30min at 4° C. in 1 mg/ml solution of sulfo-NHS-SS in phosphate-bufferedsaline. Excess biotin reagent is removed by washing the cells withTris-buffered saline. The biotinylated cells are then reacted with thebiotinylated antibody receiver in the presence of streptavidin to formthe synthetic membrane-receiver complex.

In another embodiment, the receiver may be attached to the surface of,e.g., an erythroid cell or platelet with a bispecific antibody togenerate the synthetic membrane-receiver complex. For example, thebispecific antibody can have specificity for the erythroid cell orplatelet and the receiver.

In another embodiment, the receiver is attached to, e.g., an erythroidcell or platelet via a covalent attachment to generate a syntheticmembrane-receiver complex. For example, the receiver may be derivatizedand bound to the erythroid cell or platelet using a coupling compoundcontaining an electrophilic group that will react with nucleophiles onthe erythroid cell or platelet to form the interbonded relationship.Representative of these electrophilic groups are α,β unsaturatedcarbonyls, alkyl halides and thiol reagents such as substitutedmaleimides. In addition, the coupling compound can be coupled to areceiver polypeptide via one or more of the functional groups in thepolypeptide such as amino, carboxyl and tryosine groups. For thispurpose, coupling compounds should contain free carboxyl groups, freeamino groups, aromatic amino groups, and other groups capable ofreaction with enzyme functional groups. Highly charged receivers canalso be prepared for immobilization on, e.g., erythroid cells orplatelets through electrostatic bonding to generate syntheticmembrane-receiver complexes. Examples of these derivatives would includepolylysyl and polyglutamyl enzymes.

The choice of the reactive group embodied in the derivative depends onthe reactive conditions employed to couple the electrophile with thenucleophilic groups on the erythroid cell or platelet forimmobilization. A controlling factor is the desire not to inactivate thecoupling agent prior to coupling of the receiver immobilized by theattachment to the erythroid cell or platelet. Such couplingimmobilization reactions can proceed in a number of ways. Typically, acoupling agent can be used to form a bridge between the receiver and theerythroid cell or platelet. In this case, the coupling agent shouldpossess a functional group such as a carboxyl group which can be causedto react with the receiver. One way of preparing the receiver forconjugation includes the utilization of carboxyl groups in the couplingagent to form mixed anhydrides which react with the receiver, in whichuse is made of an activator which is capable of forming the mixedanhydride. Representative of such activators are isobutylchloroformateor other chloroformates which give a mixed anhydride with couplingagents such as 5,5′-(dithiobis(2-nitrobenzoic acid) (DTNB),p-chloromercuribenzoate (CMB), or m-maleimidobenzoic acid (MBA). Themixed anhydride of the coupling agent reacts with the receiver to yieldthe reactive derivative which in turn can react with nucleophilic groupson the erythroid cell or platelet to immobilize the receiver.

Functional groups on a receiver polypeptide, such as carboxyl groups canbe activated with carbodiimides and the like activators. Subsequently,functional groups on the bridging reagent, such as amino groups, willreact with the activated group on the receiver polypeptide to form thereactive derivative. In addition, the coupling agent should possess asecond reactive group which will react with appropriate nucleophilicgroups on the erythroid cell or platelet to form the bridge. Typical ofsuch reactive groups are alkylating agents such as iodoacetic acid, a,(3 unsaturated carbonyl compounds, such as acrylic acid and the like,thiol reagents, such as mercurials, substituted maleimides and the like.

Alternatively, functional groups on the receiver can be activated so asto react directly with nucleophiles on, e.g., erythroid cells orplatelets to obviate the need for a bridge-forming compound. For thispurpose, use is made of an activator such as Woodward's Reagent K or thelike reagent which brings about the formation of carboxyl groups in thereceiver into enol esters, as distinguished from mixed anhydrides. Theenol ester derivatives of receivers subsequently react with nucleophilicgroups on, e.g., an erythroid cell or platelet to effect immobilizationof the receiver, thereby creating a synthetic membrane-receiver complex.

In some embodiments, the synthetic membrane-receiver complex isgenerated by contacting an erythroid cell with a receiver and optionallya payload, wherein contacting does not include conjugating the receiverto the erythroid cell using an attachment site comprising Band 3(CD233), aquaporin-1, Glut-1, Kidd antigen, RhAg/Rli50 (CD241), Rli(CD240), Rh30CE (CD240CE), Rh30D (CD240D), Kx, glycophorin B (CD235b),glycophorin C (CD235c), glycophorin D (CD235d), Kell (CD238),Duffy/DARCi (CD234), CR1 (CD35), DAF (CD55), Globoside, CD44, ICAM-4(CD242), Lu/B-CAM (CD239), XG1/XG2 (CD99), EMMPRIN/neurothelin (CD147),JMH, Glycosyltransferase, Cartwright, Dombrock, C4A/CAB, Scianna, MER2,stomatin, BA-1 (CD24), GPIV (CD36), CD108, CD139, or H antigen (CD173).

In some embodiments, the synthetic membrane-receiver complex comprises areceiver that is enzymatically conjugated.

In specific embodiments, the receiver can be conjugated to the surfaceof, e.g., an erythroid cell or platelet by various chemical andenzymatic means, including but not limited to those listed in table 9 togenerate a synthetic membrane-receiver complex. These methods includechemical conjugation with bifunctional cross-linking agents such as,e.g., an NHS ester-maleimide heterobifunctional crosslinker to connect aprimary amine group with a reduced thiol group. These methods alsoinclude enzymatic strategies such as, e.g., transpeptidase reactionmediated by a sortase enzyme to connect one polypeptide containing theacceptor sequence LPXTG (SEQ ID NO: 25) or LPXTA (SEQ ID NO: 26) with apolypeptide containing the N-terminal donor sequence GGG, see e.g., Sweeet al., PNAS 2013. The methods also include combination methods, such ase.g., sortase-mediated conjugation of Click Chemistry handles (an azideand an alkyne) on the antigen and the cell, respectively, followed by acyclo-addition reaction to chemically bond the antigen to the cell, seee.g., Neves et al., Bioconjugate Chemistry, 2013.

If desired, a catalytic bond-forming polypeptide domain can be expressedon or in e.g., an erythroid cell or platelet, either intracellularly orextracellularly. Many catalytic bond-forming polypeptides exist,including transpeptidases, sortases, and isopeptidases, including thosederived from Spy0128, a protein isolated from Streptococcus pyogenes.

It has been demonstrated that splitting the autocatalytic isopeptidebond-forming subunit (CnaB2 domain) of Spy0128 results in two distinctpolypeptides that retain catalytic activity with specificity for eachother. The polypeptides in this system are termed SpyTag and SpyCatcher.Upon mixing, SpyTag and SpyCatcher undergo isopeptide bond formationbetween Asp117 on SpyTag and Lys31 on SpyCatcher (Zakeri and Howarth,JACS 2010, 132:4526). The reaction is compatible with the cellularenvironment and highly specific for protein/peptide conjugation (Zakeri,B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy,V. T.; Howarth, M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E690-E697).SpyTag and SpyCatcher has been shown to direct post-translationaltopological modification in elastin-like protein. For example, placementof SpyTag at the N-terminus and SpyCatcher at the C-terminus directsformation of circular elastin-like proteins (Zhang et al, Journal of theAmerican Chemical Society, 2013).

The components SpyTag and SpyCatcher can be interchanged such that asystem in which molecule A is fused to SpyTag and molecule B is fused toSpyCatcher is functionally equivalent to a system in which molecule A isfused to SpyCatcher and molecule B is fused to SpyTag. For the purposesof this document, when SpyTag and SpyCatcher are used, it is to beunderstood that the complementary molecule could be substituted in itsplace.

A catalytic bond-forming polypeptide, such as a SpyTag/SpyCatchersystem, can be used to attach the receiver to the surface of, e.g., anerythroid cell, to generate a synthetic membrane-receiver complex. TheSpyTag polypeptide sequence can be expressed on the extracellularsurface of the erythroid cell. The SpyTag polypeptide can be, forexample, fused to the N terminus of a type-1 or type-3 transmembraneprotein, e.g., glycophorin A, fused to the C terminus of a type-2transmembrane protein, e.g., Kell, inserted in-frame at theextracellular terminus or in an extracellular loop of a multi-passtransmembrane protein, e.g., Band 3, fused to a GPI-acceptorpolypeptide, e.g., CD55 or CD59, fused to a lipid-chain-anchoredpolypeptide, or fused to a peripheral membrane protein. The nucleic acidsequence encoding the SpyTag fusion can be expressed within a syntheticmembrane-receiver complex. A receiver polypeptide can be fused toSpyCatcher. The nucleic acid sequence encoding the SpyCatcher fusion canbe expressed and secreted from the same erythroid cell that expressesthe SpyTag fusion. Alternatively, the nucleic acid sequence encoding theSpyCatcher fusion can be produced exogenously, for example in abacterial, fungal, insect, mammalian, or cell-free production system.Upon reaction of the SpyTag and SpyCatcher polypeptides, a covalent bondwill be formed that attaches the receiver to the surface of theerythroid cell to form a synthetic membrane-receiver complex. Anerythroid cell comprising the receiver polypeptide fusion is an exampleof a synthetic membrane-receiver polypeptide complex that comprises aconjugated receiver.

In one embodiment, the SpyTag polypeptide may be expressed as a fusionto the N terminus of glycophorin A under the control of the Gatalpromoter in an erythroid cell. A receiver polypeptide, for examplecomplement receptor 1 and the receivers listed in table 7, fused to theSpyCatcher polypeptide sequence can be expressed under the control ofthe Gatal promoter in the same erythroid cell. Upon expression of bothfusion polypeptides, an isopeptide bond will be formed between theSpyTag and SpyCatcher polypeptides, forming a covalent bond between theerythroid cell surface and the receiver polypeptide. An erythroid cellcomprising the receiver polypeptide fusion is an example of a syntheticmembrane-receiver polypeptide complex that comprises a conjugatedreceiver.

In another embodiment, the SpyTag polypeptide may be expressed as afusion to the N terminus of glycophorin A under the control of the Gatalpromoter in an erythroid cell. A receiver polypeptide, for examplecomplement receptor 1, fused to the SpyCatcher polypeptide sequence canbe expressed in a suitable mammalian cell expression system, for exampleHEK293 cells. Upon expression of the SpyTag fusion polypeptide on theerythroid cell, the SpyCatcher fusion polypeptide can be brought incontact with the cell. Under suitable reaction conditions, an isopeptidebond will be formed between the SpyTag and SpyCatcher polypeptides,forming a covalent bond between the erythroid cell surface and thereceiver polypeptide. An erythroid cell comprising the receiverpolypeptide fusion is an example of a synthetic membrane-receiverpolypeptide complex that comprises a conjugated receiver.

A catalytic bond-forming polypeptide, such as a SpyTag/SpyCatchersystem, can be used to anchor a receiver molecule to the intracellularspace of an erythroid cell. The SpyTag polypeptide sequence can beexpressed in the intracellular space of the erythroid cell by a numberof methods, including direct expression of the transgene, fusion to anendogenous intracellular protein such as, e.g., hemoglobin, fusion tothe intracellular domain of endogenous cell surface proteins such as,e.g., Band 3, glycophorin A, Kell, or fusion to a structural componentof the erythroid cytoskeleton. The SpyTag sequence is not limited to apolypeptide terminus and may be integrated within the interior sequenceof an endogenous polypeptide such that polypeptide translation andlocalization is not perturbed. A receiver polypeptide can be fused toSpyCatcher. The nucleic acid sequence encoding the SpyCatcher fusion canbe expressed within the same erythroid cell that expresses the SpyTagfusion. Upon reaction of the SpyTag and SpyCatcher polypeptides, acovalent bond will be formed that acts to anchor the receiverpolypeptide in the intracellular space of the erythroid cell. Anerythroid cell comprising the receiver polypeptide fusion is an exampleof a synthetic membrane-receiver polypeptide complex that comprises aconjugated receiver.

In one embodiment, an erythroid cell may express SpyTag fused tohemoglobin beta intracellularly. The erythroid cell may be geneticallymodified with a gene sequence that includes a hemoglobin promoter, betaglobin gene and a SpyTag sequence such that upon translation,intracellular beta globin is fused to SpyTag at is C terminus. Inaddition, the erythroid cell expresses a Gatal promoter-led gene thatcodes for SpyCatcher driving phenylalanine hydroxylase (PAH) expressionsuch that upon translation, intracellular PAH is fused to SpyCatcher atits N terminus. Upon expression of both fusion proteins the SpyTag boundbeta globin is linked through an isopeptide bond to the SpyCatcher boundPAH in the intracellular space, allowing PAH to be anchored to betaglobin and retained during maturation. An erythroid cell comprising thereceiver polypeptide fusion is an example of a syntheticmembrane-receiver polypeptide complex that comprises a conjugatedreceiver.

In another embodiment, the SpyTag polypeptide can be expressed as afusion to the receiver polypeptide within an erythroid cell. TheSpyCatcher polypeptide can be expressed as a fusion to the C terminus(intracellular) of glycophorin A within the same erythroid cell. Uponexpression of both fusion polypeptides, an isopeptide bond will beformed between the SpyTag and SpyCatcher polypeptides, forming acovalent bond between the membrane-anchored endogenous erythroidpolypeptide and the receiver molecule. An erythroid cell comprising thereceiver polypeptide fusion is an example of a syntheticmembrane-receiver polypeptide complex that comprises a conjugatedreceiver.

Other molecular fusions may be formed between polypeptides and includedirect or indirect conjugation. The polypeptides may be directlyconjugated to each other or indirectly through a linker. The linker maybe a peptide, a polymer, an aptamer, or a nucleic acid. The polymer maybe, e.g., natural, synthetic, linear, or branched. Receiver polypeptidescan comprise a heterologous fusion protein that comprises a firstpolypeptide and a second polypeptide with the fusion protein comprisingthe polypeptides directly joined to each other or with interveninglinker sequences and/or further sequences at one or both ends. Theconjugation to the linker may be through covalent bonds or ionic bonds.

In certain embodiments, the polypeptide receiver is loaded into thesynthetic membrane-receiver polypeptide complex. Non-polypeptidereceivers may also be loaded within a synthetic membrane-receivercomplex. In some embodiments, synthetic membrane-receiver complexes aregenerated by loading, e.g., erythroid cells or platelets with one ormore receivers, such that the one or more receivers are internalizedwithin the erythroid cells or platelets. Optionally, the erythroid cellsor platelets may additionally be loaded with a payload, such as, e.g., atherapeutic agent.

A number of methods may be used to load, e.g., erythroid cells orplatelets with a receiver and optionally a payload (e.g., a therapeuticagent). Suitable methods include, for example, hypotonic lysis,hypotonic dialysis, osmosis, osmotic pulsing, osmotic shock,ionophoresis, electroporation, sonication, microinjection, calciumprecipitation, membrane intercalation, lipid mediated transfection,detergent treatment, viral infection, diffusion, receptor mediatedendocytosis, use of protein transduction domains, particle firing,membrane fusion, freeze-thawing, mechanical disruption, and filtration.Any one such method or a combination thereof may be used to generate thesynthetic membrane-receiver complexes described herein.

For hypotonic lysis, e.g., erythroid cell are exposed to low ionicstrength buffer causing them to burst. The receiver or the payload(e.g., a therapeutic agent) distributes within the cells. Erythroidcell, specifically red blood cells may be hypotonically lysed by adding30-50 fold volume excess of 5 mM phosphate buffer (pH 8) to a pellet ofisolated red blood cells. The resulting lysed cell membranes areisolated by centrifugation. The pellet of lysed red blood cell membranesis resuspended and incubated in the presence of the receiver and/ortherapeutic agent in a low ionic strength buffer, e.g., for 30 min.Alternatively, the lysed red blood cell membranes may be incubated withthe receiver or the payload (e.g., a therapeutic agent) for as little asone minute or as long as several days, depending upon the bestconditions determined to efficiently load the erythroid cells.

Alternatively, erythroid cells, specifically red blood cells may beloaded with a receiver and optionally a payload (e.g., a therapeuticagent) using controlled dialysis against a hypotonic solution to swellthe cells and create pores in the cell membrane (See, e.g., U.S. Pat.Nos. 4,327,710; 5,753,221; and 6,495,351). For example, a pellet ofisolated red blood cells is resuspended in 10 mM HEPES, 140 mM NaCl, 5mM glucose pH 7.4 and dialyzed against a low ionic strength buffercontaining 10 mM NaH₂PO₄, 10 mM NaHCO₃, 20 mM glucose, and 4 mM MgCl₂,pH 7.4. After 30-60 min, the red blood cells are further dialyzedagainst 16 mM NaH₂PO₄, pH 7.4 solution containing the receiver or thepayload (e.g., a therapeutic agent) for an additional 30-60 min. All ofthese procedures may be advantageously performed at a temperature of 4°C. In some instances, it may be beneficial to load a large quantity oferythroid cells, specifically red blood cells with a therapeutic agentby a dialysis approach and a specific apparatus designed for thispurpose may be used (See, e.g., U.S. Pat. Nos. 4,327,710, 6,139,836 and6,495,351 B2).

The loaded erythroid cells, specifically red blood cells can be resealedby gentle heating in the presence of a physiological solution such as,for example, 0.9% saline, phosphate buffered saline, Ringer's solution,cell culture medium, blood plasma or lymphatic fluid. For example,well-sealed membranes may be generated by treating the disruptederythroid cells, specifically red blood cells for 1-2 min in 150 mM saltsolution of, for example, 100 mM phosphate (pH 8.0) and 150 mM sodiumchloride at a temperature of 60° C. Alternatively, the cells may beincubated at a temperature of 25-50° C. for 30 min to 4 h (See, e.g.,U.S. Patent Application 2007/0243137 A1). Alternatively, the disruptedred blood cells may be resealed by incubation in 5 mM adenine, 100 mMinosine, 2 mM ATP, 100 mM glucose, 100 mM Na-pyruvate, 4 mM MgCl2, 194mM NaCl, 1.6 M KCl, and 35 mM NaH₂PO₄, pH 7.4 at a temperature of 37° C.for 20-30 min (See, e.g., U.S. Pat. No. 5,753,221).

For electroporation, e.g., erythroid cells or platelets are exposed toan electrical field which causes transient holes in the cell membrane,allowing the receiver and optional payload (e.g., therapeutic agent) todiffuse into the cell (See, e.g., U.S. Pat. No. 4,935,223). Erythroidcells, specifically red blood cells, for example, are suspended in aphysiological and electrically conductive media such as platelet-freeplasma to which the receiver and optional payload (e.g., therapeuticagent) is added. The mixture in a volume ranging from 0.2 to 1.0 ml isplaced in an electroporation cuvette and cooled on ice for 10 min. Thecuvette is placed in an electroporation apparatus such as, for example,an ECM 830 (from BTX Instrument Division, Harvard Apparatus, Holliston,Mass.). The cells are electroporated with a single pulse ofapproximately 2.4 milliseconds in length and a field strength ofapproximately 2.0 kV/cm. Alternatively, electroporation of erythroidcells, specifically red blood cells may be carried out using doublepulses of 2.2 kV delivered at 0.25 μF using a Bio-Rad Gene Pulsarapparatus (Bio-Rad, Hercules, Calif., USA) to achieve a loading capacityof over 60% (Flynn et al., Cancer Lett. 82:225-229 (1994)). The cuvetteis returned to the ice bath for 10-60 min and then placed in a 37° C.water bath to induce resealing of the cell membrane. Any suitableelectroporation method may be used to generate the syntheticmembrane-receiver complexes described herein.

For sonication, erythroid cells are, for example, exposed to highintensity sound waves, causing transient disruption of the cell membraneallowing the receiver and optional payload (e.g., therapeutic agent) todiffuse into the cell. Any suitable sonication method may be used togenerate the synthetic membrane-receiver complexes described herein.

For detergent treatment, erythroid cells, for example, are treated witha mild detergent which transiently compromises the cell membrane bycreating holes through which the receiver and optional payload (e.g.,therapeutic agent) may diffuse. After cells are loaded, the detergent iswashed from the cells. For example, the detergent may be saponin. Anysuitable detergent treatment method may be used to generate thesynthetic membrane-receiver complexes described herein.

For receptor mediated endocytosis, erythroid cells, for example, mayhave a surface receptor which upon binding of the receiver or payload(e.g., therapeutic agent) induces internalization of the receptor andthe associated receiver or payload (e.g., therapeutic agent). Anysuitable endocytosis method may be used to generate the syntheticmembrane-receiver complexes described herein.

In some embodiments, the receiver and optional payload (e.g.,therapeutic agent) may be loaded, e.g., into an erythroid cell orplatelet by fusing or conjugating the receiver or payload to proteinsand/or polypeptides capable of crossing or translocating the plasmamembrane (See, e.g., U.S. Patent Application 2002/0151004 A1). Examplesof protein domains and sequences that are capable of translocating acell membrane include, for example, sequences from theHIV-1-transactivating protein (TAT), the Drosophila Antennapediahomeodomain protein, the herpes simplex-1 virus VP22 protein, andtransportin, a fusion between the neuropeptide galanin and the waspvenom peptide mastoparan. For example, a payload may be fused orconjugated to all or part of the TAT peptide. A receiver fusion proteincontaining all or part of the TAT peptide and/or a fusion proteincontaining all or part of the TAT peptide and the payload (e.g., atherapeutic agent, such as an antibody, enzyme, or peptide) may begenerated using standard recombinant DNA methods. Alternatively, all orpart of the TAT peptide (including receivers comprising all or part ofthe TAT peptide) may be chemically coupled to a functional groupassociated with the payload (e.g., therapeutic agent) such as, forexample, a hydroxyl, carboxyl or amino group. In some instances, thelink between the TAT peptide and the payload may be pH sensitive suchthat once the conjugate or fusion has entered the intracellularenvironment, the therapeutic agent is separated from the TAT peptide.

In some embodiments, the synthetic membrane-receiver complex isgenerated by contacting an erythroid cell with a receiver and optionallya payload without lysing and resealing the cells to incorporate thereceiver and/or payload. In some embodiments, the syntheticmembrane-receiver complex is generated by contacting an erythroid cellwith a receiver and optionally a payload, wherein contacting does notcomprise hypotonic dialysis.

In some embodiments, the synthetic membrane-receiver complex isgenerated by contacting an erythroid cell with a receiver and optionallya payload, wherein contacting does not include loading the receiverand/or payload into or onto the erythroid cell. In some embodiments, thereceiver is generated in an entity that is not the erythroid cell to becontacted and/or the receiver is isolated from a sample that does notcomprise the erythroid cell to be contacted. For example, for apolypeptide receiver suitable entities include a cell line, an in vitroexpression system, a bacterial expression system, etc.

For mechanical firing, erythroid cells, for example, may be bombardedwith the receiver and optional payload (e.g., therapeutic agent)attached to a heavy or charged particle such as, for example, goldmicrocarriers and are mechanically or electrically accelerated such thatthey traverse the cell membrane. Microparticle bombardment may beachieved using, for example, the Helios Gene Gun (from, e.g., Bio-Rad,Hercules, Calif., USA). Any suitable microparticle bombardment methodmay be used to generate the synthetic membrane-receiver complexesdescribed herein.

In some embodiments, erythroid cells or platelets may be loaded with areceiver and optional payload (e.g., therapeutic agent) by fusion with asynthetic vesicle such as, for example, a liposome. In this instance,the vesicles themselves are loaded with the receiver and optionalpayload using one or more of the methods described herein or known inthe art. Alternatively, the receiver and optional payload (e.g.,therapeutic agent) may be loaded into the vesicles during vesicleformation. The loaded vesicles are then fused with the erythroid cellsor platelets under conditions that enhance cell fusion. Fusion of aliposome, for example, with a cell may be facilitated using variousinducing agents such as, for example, proteins, peptides, polyethyleneglycol (PEG), and viral envelope proteins or by changes in mediumconditions such as pH (See, e.g., U.S. Pat. No. 5,677,176). Any suitableliposomal fusion method may be used to generate the syntheticmembrane-receiver complexes described herein.

For filtration, erythroid cells or platelets and the receiver andoptional payload (e.g., therapeutic agent) may be forced through afilter of pore size smaller than the cell causing transient disruptionof the cell membrane and allowing the receiver and optional therapeuticagent to enter the cell. Any suitable filtration method may be used togenerate the synthetic membrane-receiver complexes described herein.

For freeze thawing, erythroid cells are subjected to several freeze thawcycles, resulting in cell membrane disruption (See, e.g., U.S. PatentApplication 2007/0243137 A1). In this instance, a pellet of packed redblood cells (0.1-1.0 ml) is mixed with an equal volume (0.1-1.0 ml) ofan isotonic solution (e.g., phosphate buffered saline) containing thereceiver and optional payload (e.g., therapeutic agent). The red bloodcells are frozen by immersing the tube containing the cells and receiverand optional payload into liquid nitrogen. Alternatively, the cells maybe frozen by placing the tube in a freezer at −20° C. or −80° C. Thecells are then thawed in, e.g., a 23° C. water bath and the cyclerepeated if necessary to increase loading. Any suitable freeze-thawmethod may be used to generate the synthetic membrane-receiver complexesdescribed herein.

The receiver and optional payload (e.g., therapeutic agent) may beloaded into a cell, e.g., an erythroid cell or platelet in a solubilizedform, e.g., solubilized in an appropriate buffer prior to loading intoerythroid cells or platelets.

Alternatively, the receiver and optional payload (e.g., therapeuticagent) may be loaded into a cell, e.g., an erythroid cell or platelet ina particulate form as a solid microparticulate (See, e.g., U.S. PatentApplications 2005/0276861 A1 and U.S. 2006/0270030 A1). In thisinstance, the receiver or payload may be poorly water-soluble with asolubility of less than 1-10 mg/ml. Microparticles of poorlywater-soluble receivers or payloads can be made of less than 10 μm usinga variety of techniques such as, for example, energy addition techniquessuch as milling (e.g., pearl milling, ball milling, hammer milling,fluid energy milling, jet milling), wet grinding, cavitation or shearingwith a microfluidizer, and sonication; precipitation techniques such as,for example, microprecipitation, emulsion precipitation,solvent-antisolvent precipitation, phase inversion precipitation, pHshift precipitation, infusion precipitation, temperature shiftprecipitation, solvent evaporation precipitation, reactionprecipitation, compressed fluid precipitation, protein microsphereprecipitation; and other techniques such as spraying into cryogenicfluids (See, e.g., U.S. Patent Application 2005/0276861 A1). Watersoluble receivers or payloads may also be used to form solidmicroparticles in the presence of various polymers such as, for example,polylactate-polyglycolate copolymer (PLGA), polycyanoacrylate, albumin,and/or starch (See, e.g., U.S. Patent Application 2005/0276861 A1).Alternatively, a water soluble receivers or payloads may be encapsulatedin a vesicle to form a microparticle. The microparticles composed of thereceiver and optional payload (e.g., therapeutic agent) may beincorporated into a cell, such as an erythroid cell or platelet usingthe methods described herein.

In specific embodiments, synthetic membrane-receiver complexes aregenerated from erythrocytes. For example, erythrocytes may be loadedwith a receiver polypeptide or mRNA encoding a receiver polypeptide bycontrolled cell injury. The cell injury can be caused by, for example,pressure induced by mechanical strain or shear forces, subjecting thecell to deformation, constriction, rapid stretching, rapid compression,or pulse of high shear rate. The controlled cell injury leads to uptakeof material, e.g., a receiver and optionally a payload into thecytoplasm of the cell from the surrounding cell medium. Any suitablecontrolled injury method may be used to generate the syntheticmembrane-receiver complexes described herein.

Using controlled cell injury based on controlled cell deformation (e.g.,mechanical deformation of the cell as it passes through theconstriction) leads to uptake of material, e.g., a receiver andoptionally a payload by diffusion rather than endocytosis. The material,e.g., a receiver and optionally a payload is present in the cytoplasmrather than in endosomes following cellular uptake upon the controlledinjury thereby making the material readily available to the cell.Controlled cell injury, e.g., by controlled deformation, preserves cellviability (e.g., greater than 50%, 70%, or greater than 90%). In certainembodiments, controlled cell injury, e.g., by controlled deformation,preserves the state of cellular differentiation and activity. Ifdesired, a combination treatment is used, e.g., controlled injury bydeformation followed by or preceded by, e.g., electroporation or anothercell membrane permeability increasing method. Optionally, surfactantsmay be used.

Mechanical deformation methods are particularly suitable for cells thatdo not tolerate other membrane permeability increasing methods well,e.g., show decreased viability or a different state of differentiationafter performing such methods. Mechanical deformation methods are alsosuitable for material, e.g., a receiver and optionally a payload thatdoes not tolerate other membrane permeability increasing methods well.Alternatively or in addition, the receiver or payload may not besufficiently introduced into the cell using alternative methods, e.g.,because of e.g., charge, hydrophobicity, or size of the payload.

One exemplar method of controlled injury by deformation and devicessuitable for such methods is described, e.g., in PCT Publication No.WO2013059343 INTRACELLULAR DELIVERY, incorporated herein by reference.

In a specific embodiment, a population of reticulocytes is provided thathas been subjected to controlled cell injury by controlled deformationto introduce a receiver, thereby generating a syntheticmembrane-receiver complex. The cells can, e.g., be compressed anddeformed by passage through a micro-channel having a diameter less thanthat of an individual reticulocyte, thereby causing perturbations in thecell membrane such that the membrane becomes porous. Cells are moved,e.g., pushed, through the channels or conduits by application ofpressure. The compression and deformation occurs in a delivery mediumcomprising, e.g., receiver polypeptide or oligonucleotide (e.g., DNA,RNA, such as mRNA) and optionally a payload. For example, the deliverymedium may comprise a receiver including but not limited to those listedin table 7 or coding mRNA thereof. Upon deformation the reticulocytetakes up and retains the exogenous material. Following controlled injuryto the cell by constriction, stretching, and/or a pulse of high shearrate, the cells are optionally incubated in a delivery medium thatcontains the material, e.g., a receiver and optionally a payload. Thecells may be maintained in the delivery medium for a few minutes torecover, e.g., to close the injury caused by passing through theconstriction. This may occur at room temperature.

Controlled cell injury as used herein includes: i) virus-mediatedtransfection (e.g., Herpes simplex virus, Adeno virus, Adeno-associatedvirus, Vaccinia virus, or Sindbis virus), ii) chemically-mediatedtransfection, e.g., cationic polymer, calcium phosphate, cationic lipid,polymers, and nanoparticles, such as cyclodextrin, liposomes, cationicliposomes, DEAE-dextran, polyethyleneimine, dendrimer, polybrene,calcium phosphate, lipofectin, DOTAP, lipofectamine, CTAB/DOPE, DOTMA;and iii) physically-mediated transfection, including direct injection,biolistic particle delivery, electroporation, laser-irradiation,sonoporation, magnetic nanoparticles, and controlled deformation (e.g.,cell squeezing), as exemplified by micro-needle, nano-needle,femtosyringe, atomic-force microscopy (AFM) tip, gene gun (e.g., goldnanoparticles), Amaxa Nucleofector, phototransfection (multi-photonlaser), impalefection, and magnetofection, and other suitable methodsknown in the art. Any suitable method may be used to obtain a syntheticmembrane-receiver complex described herein comprising one or more DNA,RNA (e.g., mRNA encoding a receiver polypeptide), or receiverpolypeptides and optionally a payload (e.g., a therapeutic agent).

Polypeptide receivers can be detected on the synthetic membrane-receivercomplex. The presence of the receiver polypeptide can be validated andquantified using standard molecular biology methods, e.g., Westernblotting or FACS analysis. Receiver polypeptides present in theintracellular environment may be quantified upon cell lysis or usingfluorescent detection.

For example, a population of erythroid cells is loaded with adenosinedeaminase (ADA) using the Pro-Ject protein transfection reagent kit togenerate a synthetic membrane-ADA receiver complex. The population ofsynthetic membrane-ADA receiver complexes is then characterized foractive enzyme loading using LCMS to quantify adenosine and inosine.

Alternatively, the population of erythroid cells is incubated in asolution of 10 mM, 100 mM, 500 mM chlorpromazine and 0.01, 0.1, 1.0, 10,100 mg/ml of adenosine deaminase (ADA). The population of syntheticmembrane-ADA receiver complexes are then washed and fluorescent imagingis used to quantify ADA loading.

In one embodiment, a population of erythrocytes is incubated in ahypotonic salt solution containing a concentration of 0.01, 0.1, 1.0, 10mg/ml of asparaginase to generate a synthetic membrane-asparaginasereceiver complex. The cell population is incubated for 1 hr and thenresealed by incubation in a hypertonic solution for 10 mM. Thepopulation of synthetic membrane-asparaginase receiver complexes is thenincubated in an asparagine solution for 1 hr and the asparagine andaspartate concentrations are quantified using LCMS.

To generate a synthetic membrane-thymidine phosphorylase receivercomplex, a population of erythrocytes is incubated in a PBS solutioncontaining a concentration of 0.01, 0.1, 1.0, 10 mg/ml of thymidinephosphorylase that has been fused via both the C and N termini to one ormore cell penetrating peptides, including; Penetratin, Antenapedia, TAT,SynBl, SynB3, PTD-4, PTD-5, FHV Coat-(35-49), BMV Gag-(7-25), HTLV-IIRex-(4-16), D-TAT, R9-Tat, Transportan, MAP, SBP, FBP, MPG ac, MPG(NLS),Pep-1, Pep-2, polyarginines, polylysines, (RAca)6R, (RAbu)6R, (RG)6R,(RM)6R, R10, (RA)6R, R7. Following incubation, syntheticmembrane-thymidine phosphorylase receiver complexes are placed in asolution of thymidine for 1 hr and samples are quantified for thymineand thymidine content using LCMS.

Cells may be loaded using a microfluidic device that transiently poratesthe cells, allowing a payload to enter when the cells are pressuredthrough the system. In one embodiment, a population of erythrocytes ispressured through a system of microfluidic channels in a buffer solutioncontaining 0.01, 0.1, 1.0, 10 mg/ml of phenylalanine ammoniahydroxylase. The cell suspension is then characterized for enzymaticactivity using LCMS to quantify phenylalanine and trans-cinnamic acid.

In one embodiment, a synthetic cell membrane-receiver complexes areincubated in a hypotonic solution containing 1 mM of adenosine deaminasefor 1 hr. The synthetic membrane-receiver complexes are then transferredto an isotonic solution and allowed to equilibrate and seal in thesoluble protein.

Payloads for Synthetic Membrane-Receiver Complexes

Synthetic membrane-receiver complexes may optionally be loaded withpayloads such as peptides, proteins, DNA, RNA, siRNA, and othermacromolecules and small therapeutic molecules. In some embodiments, thepayload is transferred to a cell, e.g., an erythroid cell or platelet byapplying controlled injury to the cell for a predetermined amount oftime in order to cause perturbations in the cell membrane such that thepayload can be delivered to the inside of the cell (e.g., cytoplasm).

The payload may be a therapeutic agent selected from a variety of knownsmall molecule pharmaceuticals. Alternatively, the payload may be may bea therapeutic agent selected from a variety of macromolecules, such as,e.g., an inactivating peptide nuclei acid (PNA), an RNA or DNAoligonucleotide aptamer, an interfering RNA (iRNA), a peptide, or aprotein.

In some embodiments, the synthetic membrane-receiver complex isgenerated from a reticulocyte. For example, reticulocytes may be loadedwith an mRNA encoding for a therapeutic exogenous polypeptide bycontrolled cell injury. The mRNA may be naked or modified, as desired.mRNA modification that improve mRNA stability and/or decreaseimmunogenicity include, e.g., ARCA: anti-reverse cap analog (m₂^(7.3′-O)GP₃G), GP₃G (Unmethylated Cap Analog), m⁷GP₃G (MonomethylatedCap Analog), m₃ ^(2.2.7)GP₃G (Trimethylated Cap Analog), m5CTP(5′-methyl-cytidine triphosphate), m6ATP(N6-methyl-adenosine-5′-triphosphate), s2UTP (2-thio-uridinetriphosphate), and Ψ (pseudouridine triphosphate).

Synthetic membrane-receiver complexes may comprise two or more payloads,including mixtures, fusions, combinations and conjugates, of atoms,molecules, etc. as disclosed herein, for example including but notlimited to, a nucleic acid combined with a polypeptide; two or morepolypeptides conjugated to each other; a protein conjugated to abiologically active molecule (which may be a small molecule such as aprodrug); and the like.

In some embodiments, the pharmaceutical composition comprises one ormore therapeutic agents and the synthetic membrane-receiver complexdescribed herein. In some embodiments, the the syntheticmembrane-receiver complexes are co-administered with of one or moreseparate therapeutic agents, wherein co-administration includesadministration of the separate therapeutic agent before, after orconcurrent with administration of the synthetic membrane-receivercomplex.

Suitable payloads include, without limitation, pharmacologically activedrugs and genetically active molecules, including antineoplastic agents,anti-inflammatory agents, hormones or hormone antagonists, ion channelmodifiers, and neuroactive agents. Examples of suitable payloads oftherapeutic agents include those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Drugs Acting on the Central NervousSystem; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions;Drugs Affecting Renal Function and Electrolyte Metabolism;Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; DrugsAffecting Uterine Motility; Chemotherapy of Parasitic Infections;Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases;Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs;Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology,all incorporated herein by reference. Suitable payloads further includetoxins, and biological and chemical warfare agents, for example seeSomani, S. M. (ed.), Chemical Warfare Agents, Academic Press, New York(1992)).

In some embodiments, the synthetic membrane-receiver complex does notcomprise a payload comprising a synthetic triphosphorylated nucleosideanalog. In some embodiments, the synthetic membrane-receiver complexdoes not comprise a payload comprising2′,3′-dideoxycytidine-5′-triphosphate (ddCTP) and/or3′-azido-3′-deoxythymidine-5′-triphosphate (AZT-TP).

In some embodiments, the synthetic membrane-receiver complex does notcomprise a payload comprising a bisphosphonate.

In some embodiments, the payload is a therapeutic agent, such as a smallmolecule drug or a large molecule biologic. Large molecule biologicsinclude, but are not limited to, a protein, polypeptide, or peptide,including, but not limited to, a structural protein, an enzyme, acytokine (such as an interferon and/or an interleukin), a polyclonal ormonoclonal antibody, or an effective part thereof, such as an Fvfragment, which antibody or part thereof, may be natural, synthetic orhumanized, a peptide hormone, a receptor, or a signaling molecule.

Large molecule biologics are immunoglobulins, antibodies, Fv fragments,etc., that are capable of binding to antigens in an intracellularenvironment. These types of molecules are known as “intrabodies” or“intracellular antibodies.” An “intracellular antibody” or an“intrabody” includes an antibody that is capable of binding to itstarget or cognate antigen within the environment of a cell, or in anenvironment that mimics an environment within the cell. Selectionmethods for directly identifying such “intrabodies” include the use ofan in vivo two-hybrid system for selecting antibodies with the abilityto bind to antigens inside mammalian cells. Such methods are describedin PCT/GB00/00876, incorporated herein by reference. Techniques forproducing intracellular antibodies, such as anti-β-galactosidase scFvs,have also been described in Martineau et al., J Mol Biol 280:117-127(1998) and Visintin et al., Proc. Natl. Acad. Sci. USA 96:11723-1728(1999).

Large molecule biologics include but is not limited to, at least one ofa protein, a polypeptide, a peptide, a nucleic acid, a virus, avirus-like particle, an amino acid, an amino acid analogue, a modifiedamino acid, a modified amino acid analogue, a steroid, a proteoglycan, alipid and a carbohydrate or a combination thereof (e.g., chromosomalmaterial comprising both protein and DNA components or a pair or set ofeffectors, wherein one or more convert another to active form, forexample catalytically).

A Large molecule biologic may include a nucleic acid, including, but notlimited to, an oligonucleotide or modified oligonucleotide, an antisenseoligonucleotide or modified antisense oligonucleotide, an aptamer, acDNA, genomic DNA, an artificial or natural chromosome (e.g., a yeastartificial chromosome) or a part thereof, RNA, including an siRNA, ashRNA, mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA);a virus or virus-like particles; a nucleotide or ribonucleotide orsynthetic analogue thereof, which may be modified or unmodified.

The large molecule biologic can also be an amino acid or analoguethereof, which may be modified or unmodified or a non-peptide (e.g.,steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. If thelarge molecule biologic is a polypeptide, it can be loaded directlyinto, e.g., an erythroid cell or a platelet according to the methodsdescribed herein. Alternatively, an exogenous nucleic acid encoding apolypeptide, which sequence is operatively linked to transcriptional andtranslational regulatory elements active in a cell at a target site, maybe loaded.

Small molecules, including inorganic and organic chemicals, may also beused as payloads of the synthetic membrane-receiver complexes describedherein.

In some embodiments, the small molecule is a pharmaceutically activeagent. Useful classes of pharmaceutically active agents include, but arenot limited to, antibiotics, anti-inflammatory drugs, angiogenic orvasoactive agents, growth factors and chemotherapeutic (anti-neoplastic)agents (e.g., tumour suppressers).

If a prodrug is loaded into the synthetic membrane-receiver complex inan inactive form it is often useful that the synthetic membrane-receivercomplex further comprises a receiver such as an activating polypeptidewhich converts the inactive prodrug to active drug form. In anembodiment, activating receiver polypeptides include, but are notlimited to, viral thymidine kinase (encoded by Genbank Accession No.J02224), carboxypeptidase A (encoded by Genbank Accession No. M27717),α-galactosidase (encoded by Genbank Accession No. M13571),β-gluucuronidase (encoded by Genbank Accession No. M15182), alkalinephosphatase (encoded by Genbank Accession No. J03252 J03512), orcytochrome P-450 (encoded by Genbank Accession No. D00003 N00003),plasmin, carboxypeptidase G2, cytosine deaminase, glucose oxidase,xanthine oxidase, β-glucosidase, azoreductase, t-gutamyl transferase,β-lactamase, and penicillin amidase.

Either the receiver polypeptide or the exogenous gene encoding it may beloaded into, e.g., an erythroid cell or platelet, to generate asynthetic membrane-receiver complex. Both the prodrug and the activatingreceiver polypeptide may be encoded by genes on the same exogenousnucleic acid. Furthermore, either the prodrug or the the activatingreceiver polypeptide of the prodrug may be transgenically expressed in asynthetic membrane-receiver complex.

The synthetic membrane-receiver complexes may also be labeled with oneor more positive markers that can be used to monitor over time thenumber or concentration of synthetic membrane-receiver complexes in theblood circulation of an individual. The overall number of syntheticmembrane-receiver complexes will decay over time following initialtransfusion. In some embodiments, the signal from one or more positivemarkers are correlated with that of an activated molecular marker,generating a proportionality of signal that is independent of the numberof synthetic membrane-receiver complexes remaining in the circulation.Suitable fluorescent compounds include those that are approved by theFood & Drug Administration for human use including but not limited tofluorescein, indocyanin green, and rhodamine B. For example, syntheticmembrane-receiver complexes may be non-specifically labeled withfluorescein isothiocyanate (FITC; Bratosin et al., Cytometry 46:351-356(2001)). For example, a solution of FITC-labeled lectins in phosphatebuffered saline (PBS) with 0.2 mM phenylmethysulfonyl fluoride (PMSF) isadded to an equal volume of isolated erythroid cells or platelets in thesame buffer. The cells are incubated with the FITC-labeled lectins for 1h at 4° C. in the dark. The lectins bind to sialic acids andbeta-galactosyl residues on the surface of the erythroid cells.

Other dyes may be useful for tracking synthetic membrane-receivercomplexes in human and non-human circulation. A number of reagents maybe used to non-specifically label a synthetic membrane-receiver complex.For example, erythroid cells or platelets may be labeled with PKH26 Red(See, e.g., Bratosin, et al., (1997) Cytometry 30:269-274). Erythroidcells or platelets (1-3×10⁷ cells) are suspended in 1 ml of diluent andrapidly added to 1 ml or 2 μM PKH26 dissolved in the same diluent. Themixture is mixed by gentle pipetting and incubated at 25° C. for 2-5 minwith constant stirring. The labeling may be stopped by adding an equalvolume of human serum or compatible protein solution (e.g., 1% bovineserum albumin). After an additional minute, an equal volume of cellculture medium is added and the cells are isolated by centrifugation at2000×g for 5 min Cells are washed three times by repeated suspension incell culture medium and centrifugation. PHK26-labeled syntheticmembrane-receiver complexes may be monitored with a maximum excitationwavelength of 551 nm and a maximum emission wavelength of 567 nm.

Synthetic membrane-receiver complexes may be tracked in vivo usingVivoTag 680 (VT680; VisEn Medical, Woburn, Mass., USA), a near-infraredfluorochrome with a peak excitation wavelength of 670±5 nm and a peakemission wavelength of 688±5 nm. VT680 also contains an amine reactiveNHS ester which enables it to cross-link with proteins and peptides. Thesurface of cells, e.g., erythroid cells or platelets may be labeled withVT680 (See, e.g., Swirski, et al., (2007) PloS ONE 10:e1075). Forexample, 4×10⁶ cells/ml are incubated with VT680 diluted in completeculture medium at a final concentration of 0.3 to 300 μg/ml for 30 minat 37° C. The cells are washed twice with complete culture medium afterlabeling. Cells may be non-specifically labeled based on proteinsexpressed on the surface of the synthetic membrane-receiver complex.Alternatively, a specific protein, such as a receiver may be labeledwith VT680. In some embodiments, a protein or peptide may be directlylabeled with VT680 ex vivo and subsequently either attached to thesurface of the cell or incorporated into the interior of the cell usingmethods described herein. In vivo monitoring may, for example, beperformed using the dorsal skin fold. Laser scanning microscopy may beperformed using, for example, an Olympus IV 100 in which VT680 isexcited with a red laser diode of 637 nm and detected with a 660/LPfilter. Alternatively, multiphoton microscopy may be performed using,for example, a BioRad Radiance 2100 MP centered around an Olympus BX51equipped with a 20×/0.95 NA objective lens and a pulsed Ti:Sapphirelaser tuned to 820 nm. The latter wavelength is chosen because VT680 hasa peak in its two-photon cross-section at 820 nm.

Alternatively or in addition, a synthetic membrane-receiver complex maybe labeled with other red and/or near-infrared dyes including, forexample, cyanine dyes such as Cy5, Cy5.5, and Cy7 (Amersham Biosciences,Piscataway, N.J., USA) and/or a variety of Alexa Fluor dyes includingAlexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 (MolecularProbes-Invitrogen, Carlsbad, Calif., USA). Additional suitablefluorophores include IRD41 and IRD700 (LI-COR, Lincoln, Nebr., USA),NIR-1 and 1C5-OSu (Dejindo, Kumamotot, Japan), LaJolla Blue (Diatron,Miami, Fla., USA), FAR-Blue, FAR-Green One, and FAR-Green Two(Innosense, Giacosa, Italy), ADS 790-NS and ADS 821-NS (American DyeSource, Montreal, Calif.). Quantum dots (Qdots) of variousemission/excitation properties may also be used for labeling syntheticmembrane-receiver complexes (See, e.g., Jaiswal et al., Nature Biotech.21:47-51 (2003)). Many of these fluorophores are available fromcommercial sources either attached to primary or secondary antibodies oras amine-reactive succinimidyl or monosuccinimidyl esters, for example,ready for conjugation to a protein or proteins either on the surface orinside the synthetic membrane-receiver complex.

Magnetic nanoparticles may be used to track synthetic membrane-receivercomplexes in vivo using high resolution MRI (Montet-Abou et al.,Molecular Imaging 4:165-171 (2005)). Magnetic particles may beinternalized by several mechanisms. Magnetic particles may be taken upby a cell, e.g., an erythroid cell or a platelet through fluid-phasepinocytosis or phagocytosis. Alternatively, the magnetic particles maybe modified to contain a surface agent such as, for example, a membranetranslocating HIV TAT peptide which promotes internalization. In someinstances, a magnetic nanoparticle such as, for example, Feridex IV®, anFDA approved magnetic resonance contrast reagent, may be internalizedinto, e.g., erythroid cells or platelets in conjunction with atransfection agent such as, for example, protamine sulfate (PRO),polylysine (PLL), and lipofectamine (LFA).

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes are generated comprising contacting an erythroid cell with areceiver, such as a polypeptide. In some embodiments, the receiverpolypeptide is encoded by an exogenous nucleic acid and is expressed bythe erythroid cell. In some embodiments, a naturally occurring erythroidcell does not comprise the receiver. For example, a naturally occurringerythroid cell does not express an endogenous polypeptide that isstructurally and functionally the same as the receiver polypeptide. Insome embodiments, the erythroid cell comprises a receiver that isover-expressed. For example, the receiver is present in substantiallyhigher copy numbers than it would be if it were endogenously expressedby a naturally occurring erythroid cell. In some embodiments, thesynthetic membrane-receiver polypeptide complexes are generated bydifferentiating and maturing the erythroid cells in vitro or in vivoafter contacting the cells with a receiver. It is known in the art thaterythrocytes undergo a complex process of maturation as theydifferentiate from precursor cells. The maturation process includes asubstantial cytoskeleton and membrane rearrangement and degradation orexpulsion of non-essential polypeptides, see e.g., Liu J et al. (2010)Blood 115(10):2021-2027; and Lodish H F et al. (1975) DevelopmentalBiology 47(1):59). For naturally occurring erythrocytes this maturationprocess happens in vivo, first in the bone marrow and then incirculation as reticulocytes mature into erythrocytes. For culturederythrocytes this maturation process happens both ex vivo, in culture,and in vivo in circulation as cultured reticulocytes mature intoeyrthrocytes (see e.g., Neildez-Nguyen et al. 2002 Nature Biotechnol20:467). In some embodiments, the synthetic membrane-receiverpolypeptide complexes generated from erythroid cells retain theirreceivers during the maturation process, in vitro or in vivo and thereceivers are not lost. In some embodiments, the syntheticmembrane-receiver polypeptide complexes generated from erythroid cellsretain their receivers after maturation. In some embodiments, fullymatured synthetic membrane-receiver polypeptide complexes generated fromerythroid cells retain their receiver. The receiver may be retained invitro, e.g., in culture and/or may be retained in vivo, e.g., afteradministration to the circulatory system of the subject. In someembodiments, the receiver may be retained by the syntheticmembrane-receiver polypeptide complexes for the life of the complex incirculation. These findings are surprising in view of the art whichsuggested that receivers would be excluded from the erythroid cellsduring the maturation process. It was further unexpected that receiverswould be retained and functionally active when the syntheticmembrane-receiver polypeptide complexes generated from erythroid cellsare administered to the circulatory system of a subject. In someembodiments culturing of eythroid cells comprising a receiver provides amethod of producing a substantially more homogeneous and/orsubstantially more scalable population of therapeutic syntheticmembrane-receiver complexes than is achievable by methods relying uponisolation and modification of non-cultured erythrocytes. Despite a greatneed for human erythroid cell-based treatment and preventive methods andrecognition for its value in the art, no systems derived from modifiedcultured cells have previously been generated or shown to retainreceiver activity in circulation, and the art suggested that suchsystems would not be achievable. When cultured human erythrocytes havebeen experimentally administered to a human subject previously they wereunmodified (Giarratana et al., Blood 2011, 118:5071).

Targets

Provided herein are synthetic membrane-receiver polypeptide complexescomprising a receiver polypeptide capable of interacting with a target.Further provided herein are synthetic membrane-receiver complexescomprising a non-polypeptide receiver capable of interacting with atarget. The synthetic membrane-receiver complexes may be administered toa subject in need thereof to modulate the amount or concentration of atarget residing in the circulatory system of the subject. A suitablereceiver may be chosen to interact with a specific target. Suitabletargets include entities that are associated with a specific disease,disorder, or condition. However, targets may also be chosen independentof a specific disease, disorder, or condition.

In some embodiments, the target is an antibody or antibody-likemolecule, for example an autoimmune or a self-antibody, or a foreignantibody, or a therapeutic antibody, including but not limited to, e.g.,an antibody against beta-2 glycoprotein 1, an antibody against I/iantigen, an antibody against the NC1 domain of collagen a3(IV), anantibody against platelet glycoprotein, an antibody againstphospholipase A2 receptor, an antibody against erythrocyte glycophorinA, B, or C, or an antibody against erythrocyte Rh antigen.

In some embodiments, the target is a molecule of the complement cascade,for example C1, C1r, C1s, C1q, C2, C2a, C2b, C3, C3a, C3b, C4, C4b, C4a,C3bBb, C3bBb3b, C4b2b, C4b2b3b, C5, C5a, C5b, C6, C7, C8, C9, poly-C9,membrane attack complex. Factor B, Factor D, Properdin, C3, C3a, C3b,iC3b, C3c, C3dg, C3dk, C3e, Bb, Factor I, C1q, C1r, C1s, C4, C4a, C4b,C2, C4 bp, Mannose-Binding Lectin (MBL), MBL-Associated Serine Protease1 (MASP1), MBL-Associated Serine Protease 2 (MASP2), C5, C5a, C6, C7,C8, C9, CR1, CR2, CR3, CR4, C3aR, C3eR, Decay-accelerating factor (DAF),Membrane cofactor protein (MCP), CD59, C3 Beta chain Receptor, C1inhibitor, C4 binding protein, Factor I, Factor H.

In some embodiments, the target is an immune complex, for example an IgGimmune complex, an IgA immune complex, an IgM immune complex.

In some embodiments, the target is an amyloid placque, for example aplacque comprised of beta amyloid, IAPP (Amylin), alpha-synuclein,PrPSc, huntingtin, calcitonin, atrial natriuretic factor, apolipoproteinAI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM.

In some embodiments, the target is a bacterium, for exampleEnterococcus, Streptococcus, or Mycobacteria, Rickettsia, Mycoplasma,Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibriocholerae, Streptococci, Staphylococcus aureus, Staphylococcusepidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae,Clostridium spp., enterotoxigenic Eschericia coli, and Bacillusanthracis. Other pathogens for which bacteremia has been reported atsome level include the following: Rickettsia, Bartonella henselae,Bartonella quintana, Coxiella burnetii, chlamydia, Mycobacterium leprae,Salmonella; shigella; Yersinia enterocolitica; Yersiniapseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis;Listeria monocytogenes; Mycoplasma spp.; Pseudomonas fluorescens; Vibriocholerae; Haemophilus influenzae; Bacillus anthracis; Treponemapallidum; Leptospira; Borrelia; Corynebacterium diphtheriae;Francisella; Brucella melitensis; Campylobacter jejuni; Enterobacter;Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

In some embodiments, the target is a virus, including but limited to,those whose infection involves injection of genetic materials into hostcells upon binding to cell surface receptors, viruses whose infection ismediated by cell surface receptors. Non-limiting examples of theseviruses can be selected from Paramyxoviridae (e.g., pneumovirus,morbillivirus, metapneumovirus, respirovirus or rubulavirus),Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., arenavirus such aslymphocytic choriomeningitis virus), Arteriviridae (e.g., porcinerespiratory and reproductive syndrome virus or equine arteritis virus),Bunyaviridae (e.g., phlebovirus or hantavirus), Caliciviridae (e.g.,Norwalk virus), Coronaviridae (e.g., coronavirus or torovirus),Filoviridae (e.g., Ebola-like viruses), Flaviviridae (e.g., hepacivirusor flavivirus), Herpesviridae (e.g., simplexvirus, varicellovirus,cytomegalovirus, roseolovirus, or lymphocryptovirus), Orthomyxoviridae(e.g., influenza virus or thogotovirus), Parvoviridae (e.g.,parvovirus), Picomaviridae (e.g., enterovirus or hepatovirus),Poxviridae (e.g., orthopoxvirus, avipoxvirus, or leporipoxvirus),Retroviridae (e.g., lentivirus or spumavirus), Reoviridae (e.g.,rotavirus), Rhabdoviridae (e.g., lyssavirus, novirhabdovirus, orvesiculovirus), and Togaviridae (e.g., alphavirus or rubivirus).Specific examples of these viruses include human respiratorycoronavirus, influenza viruses A-C, hepatitis viruses A to G, and herpessimplex viruses 1-9.

In some embodiments, the target is a parasite, including but not limitedto, for example, intestinal or blood-borne parasites, protozoa,trypanosomes; haemoprotozoa and parasites capable of causing malaria;enteric and systemic cestodes including taeniid cestodes; entericcoccidians; enteric flagellate protozoa; filarial nematodes;gastrointestinal and systemic nematodes and hookworms.

In some embodiments, the target is a fungus, including but not limitedto, for example, Candida albicans, Candida glabrata, Aspergillus, T.glabrata, Candida tropicalis, C. krusei, and C. parapsilosis.

In some embodiments, the target is a bacterial toxin, including but notlimited to, for example, AB toxin, alpha toxin, anthrax toxin,bacteriocin, botunlinum toxin, cholesterol-dependent cytolysin,Clostridium botulinum C3 toxin, Clostridium difficile toxin A,Clostridium difficile toxin B, Clostridium enterotoxin, Clostridiumperfringens alpha toxin, Clostridium perfringens beta toxin, Cordfactor, CrylAc, Cryptophycin, Delta endotoxin, Diphtheria toxin,Enterotoxin type B, erythrogenic toxin, exfoliatin, haemolysin E,heat-labile enterotoxin, heat-stable enterotoxin, hemolysin, leukocidin,lipopolysaccharide, Listeriolysin 0, microcin, Panton-Valentineleucocidin, pathogenicity island, phenol-soluble modulin, pneumolysin,pore-forming toxin, Pseudomonas exotoxin, RTX toxin, sakacin,Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin,Staphylococcus aureus delta toxin, Streptolysin, Symplocamide A,tabtoxin, tetanolysin, tetanospasmin, thiol-activated cytolysin,tolaasin, toxic shock syndrome toxin, toxoflavin, trehalose dimycolate,verocytotoxin, and vibriocin.

In some embodiments, the target is a prion protein, including but notlimited to, for example, PRP, PRPc, PRPsc, PRPres.

In some embodiments, the target is a cytokine or a chemokine or a growthfactor, including but not limited to, for example, acylation stimulatingprotein, adipokine, albinterferon, CCL1, CCL11, CCL12, CCL13, CCL14,CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL5, CCL6, CCL7, CCL8, CCL9,colony-stimulating factor, CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11,CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL6,CXCL7, CXCL9, erythropoietin, Gc-MAF, granulocyte colony-stimulatingfactor, granulocyte macrophage colony-stimulating factor, hepatocytegrowth factor, IL 10 family, IL 17 family, IL1A, IL1B, interferon,interferon beta 1a, interferon beta 1b, interferon gamma, interferontype I, interferon type II, interferon type III, interleukin,interleukin 1 family, interleukin 1 receptor antagonist, interleukin 10,interleukin 12, interleukin 12 subunit beta, interleukin 13, interleukin16, interleukin 2, interleukin 23, interleukin 23 subunit alpha,interleukin 34, interleukin 35, interleukin 6, interleukin 7,interleukin 8, interleukin-36, leukemia inhibitory factor,leukocyte-promoting factor, lymphokine, lymphotoxin, lymphotoxin alpha,lymphotoxin beta, macrophage colony-stimulating factor, macrophageinflammatory protein, macrophage-activating factor, monokine, myokine,myonectin, nicotinamide phosphoribosyltransferase, oncostatin M,oprelvekin, platelet factor 4, proinflammatory cytokine, promegapoietin,RANKL, stromal cell-derived factor 1, talimogene laherparepvec, tumornecrosis factor alpha, tumor necrosis factors, XCL1, XCL2, XCR1,angiopoietin, basic fibroblast growth factor, betacellulin, bonemorphogenetic protein, brain-derived neurotrophic factor, CCNintercellular signaling protein, CTGF, darbepoetin alfa, endoglin,epidermal growth factor, epoetin alfa, epoetin beta, erythropoietin,FGF15, FGF15/19, fibroblast growth factor, fibroblast growth factor 23,filgrastim, GLIA maturation factor, granulocyte colony-stimulatingfactor, granulocyte macrophage colony-stimulating factor, growthdifferentiation factor-9, heberprot-P, hemopoietic growth factors,heparin-binding EGF-like growth factor, hepatocyte growth factor,insulin-like growth factor, insulin-like growth factor 1, insulin-likegrowth factor 2, keratinocyte growth factor, myostatin, nerve growthfactor, neurotrophin-3, neurotrophin-4, oncomodulin, osteopromotive,palifermin, PDGFB, placental growth factor, platelet alpha-granule,platelet-derived growth factor, platelet-derived growth factor receptor,proliferative index, thrombopoietin, transforming growth factor,vascular endothelial growth factor.

In some embodiments, the target is a small molecule, for example achemical, an amino acid, an atom, an element, an organic acid, <2000 Da,<1000 Da, <500 Da, including but not limited to, for example, iron,copper, calcium, potassium, ethanol, methanol, glycine, alanine, valine,leucine, isoleucine, serine, cysteine, selenocysteine, threonine,methionine, proline, phenylalanine, tyrosine, tryptophan, histidine,lysine, arginine, aspartate, glutamate, asparagine, glutamine.

In some embodiments, the target is a lipid, lipid complex, proteolipidcomplex, or cholesterol, including but not limited to for example, LDL,VLDL, HDL, HDL2B, triglycerides, LP(a), cholesterol.

In some embodiments, the target is a mammalian cell, including but notlimited to, for example, a human cell, a circulating cell, an immunecell, a neutrophil, an eosinophil, a basophil, a lymphocyte, a monocyte,a B cell, a T cell, a CD4+ T cell, a CD8+ T cell, a gamma-delta T cell,a regulatory T cell, a natural killer cell, a natural killer T cell, amacrophage, a Kupffer cell, a dendritic cell, a cancer cell, a cancerstem cell, a circulating tumor cell, a cancer cell from one of thefollowing cancers including, but not limited to, ACUTE lymphoblasticleukaemia (ALL), ACUTE myeloid leukaemia (AML), anal cancer, bile ductcancer, bladder cancer, bone cancer, bowel cancer, brain tumours, breastcancer, cancer of unknown primary, cancer spread to bone, cancer spreadto brain, cancer spread to liver, cancer spread to lung, carcinoid,cervical cancer, choriocarcinoma, chronic lymphocytic leukaemia (CLL),chronic myeloid leukaemia (CML), colon cancer, colorectal cancer,endometrial cancer, eye cancer, gallbladder cancer, gastric cancer,gestational trophoblastic tumours (GTT), hairy cell leukaemia, head andneck cancer, hodgkin lymphoma, kidney cancer, laryngeal cancer,leukaemia, liver cancer, lung cancer, lymphoma, melanoma skin cancer,mesothelioma, men's cancer, molar pregnancy, mouth and oropharyngealcancer, myeloma, nasal and sinus cancers, nasopharyngeal cancer, nonhodgkin lymphoma (NHL), oesophageal cancer, ovarian cancer, pancreaticcancer, penile cancer, prostate cancer, rare cancers, rectal cancer,salivary gland cancer, secondary cancers, skin cancer (non melanoma),soft tissue sarcoma, stomach cancer, testicular cancer, thyroid cancer,unknown primary cancer, uterine cancer, vaginal cancer, and vulvalcancer.

Sourcing

Synthetic membrane-receiver complexes can be generated by any methoddescribed herein. In some embodiments, the steps comprise contactingisolated optionally cultured cells derived from hematopoietic stem cellswith a receiver. Hematopoietic stem cells give rise to all of the bloodcell types found in mammalian blood including myeloid (monocytes andmacrophages, neutorphils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells) and lymphoid lineages(T-cells, B-cells, NK-cells). Hematopoietic stem cells may be isolatedfrom the bone marrow of adult bones including, for example, femur, hip,rib, or sternum bones. Cells may be obtained directly from the hip, forexample, by removal of cells from the bone marrow using aspiration witha needle and syringe. Alternatively, hematopoietic stem cells may beisolated from normal peripheral blood following pre-treatment withcytokines such as, for example, granulocyte colony stimulating factor(G-CSF). G-CSF mobilizes the release of cells from the bone marrowcompartment into the peripheral circulation. Other sources ofhematopoietic stem cells include umbilical cord blood and placenta.

In some embodiments, the synthetic membrane-receiver complex isgenerated from megakaryocytes or platelets. In some embodiments, thesynthetic membrane-receiver complex is generated from an erythroid cell,such as, e.g. an erythrocyte or a reticulocyte. In some embodiments, thesynthetic membrane-receiver complex is not generated from a neutrophil,an eosinophil, or a basophil. In some embodiments, the syntheticmembrane-receiver complex is not generated from a monocyte or amacrophage.

In some embodiments, the synthetic membrane-receiver complex is notgenerated from a CD34⁺Thy-1⁺ hematopoietic stem cell or cell populationsenriched in CD34⁺Lin⁻ or CD34⁺Thy-1⁺Lin⁻ cells.

In some embodiments, the synthetic membrane-receiver complex is notgenerated from or does not comprise an autologous CD34+ cell.

Isolated hematopoietic stem cells may be cultured, expanded anddifferentiated ex vivo to provide a variety of source material togenerate synthetic membrane-receiver complexes. For example,hematopoietic stem cells isolated from bone marrow, cytokine-stimulatedperipheral blood or umbilical cord blood may be expanded anddifferentiated ex vivo into mature erythrocytes (Giarratana et al.,Nature Biotech. 23:69-74 (2005); U.S. Patent Application 2007/0218552).As such, CD34+ cells are isolated from bone marrow or peripheral or cordblood using, for example, magnetic microbead selection and Mini-MACScolumns (Miltenyi Biotech). In one example, the cells are subsequentlycultured in modified serum-free medium supplemented with 1% bovine serumalbumin (BSA), 120 μg/ml iron-saturated human transferrin, 900 ng/mlferrous sulfate, 90 ng/ml ferric nitrate and 10 μg/ml insulin andmaintained at 37° C. in 5% carbon dioxide in air. Expansion anddifferentiation of the cell culture may occur in multiple steps. Forexample, in the initial growth step following isolation, the cells maybe expanded in the medium described herein in the presence of multiplegrowth factors including, for example, hydrocortisone, stem cell factor,IL-3, and erythropoietin. In the second stage, the cells may optionallybe co-cultured, for example, on an adherent stromal layer in thepresence of erythropoietin. In a third stage, the cells may be culturedon an adherent stromal layer in culture medium in the absence ofexogenous factors. The adherent stromal layer may be murine MS-5 stromalcells, for example. Alternatively, the adherent stromal layer may bemesenchymal stromal cells derived from adult bone marrow. The adherentstromal cells may be maintained in RPMI supplemented with 10% fetal calfserum, for example. In some embodiments, the erythroid precursor cellsand cell populations derived therefrom are not co-cultured withnon-erythroid cells, e.g., with an adherent stromal layer, i.e. they arecultured in the absence of non-erythroid cells. In some embodiments,erythroid cells comprising a receiver are cultured in the absence ofnon-erythroid cells and are differentiated so that greater than 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or greater than 98% of erythroid cells are enucleated andthe population of enucleated cells is obtained without an enrichmentstep, such as gravitational separation, magnetic or fluorescent sorting,irradiation, poisoning of nucleated cells, and the like to select forenucleated cells.

In some instances, it may be desirable to expand and partiallydifferentiate the CD34+ hematopoietic stem cells in vitro and to allowterminal differentiation into mature erythrocytes to occur in vivo (See,e.g., Neildez-Nguyen et al., Nature Biotech. 20:467-472 (2002)).Isolated CD34+ hematopoietic stem cells may be expanded in vitro in theabsence of the adherent stromal cell layer in medium containing variousfactors including, for example, Flt3 ligand, stem cell factor,thrombopoietin, erythropoietin, and insulin growth factor. The resultingerythroid precursor cells may be characterized by the surface expressionof CD36 and GPA, and may be transfused into a subject where terminaldifferentiation to mature erythrocytes is allowed to occur.

In some embodiments, the erythroid cell population comprises a pluralityof enucleated functional erythroid cells that comprise a receiverpolypeptide that is retained during enucleation. The resulting isolatedenucleated functional erythroid cell comprising a receiver polypeptideexhibits substantially the same osmotic membrane fragility as acorresponding isolated, unmodified, uncultured erythroid cell.

In some embodiments, the erythroid cell population comprises a pluralityof erythrocyte precursor cells in substantially the same stage ofdifferentiation and/or cell cycle stage, wherein the precursor cellscomprise an exogenous nucleic acid encoding a receiver. The majority oferythrocyte precursor cells that comprise an exogenous nucleic acidencoding a receiver are capable of differentiating into maturefunctional erythrocytes that retain the receiver without retaining theexogenous nucleic acid.

In some embodiments, the primary cells may be collected throughvenipuncture, capillary puncture, or arterial puncture. From thecollected whole blood erythrocytes, platelets or other cells may then beisolated using one, or a combination of techniques including plasmadepletion, density gradient, Hetastarch, PrepaCyte-CB, andcentrifugation.

In some embodiments, generating a synthetic membrane-receiver complexcomprises contacting isolated optionally cultured cells that areautologous and/or allogeneic to the subject with a receiver. Forexample, erythrocytes allogeneic to the subject include one or more ofblood type specific erythrocytes or one or more universal donorerythrocytes. In some embodiments, synthetic membrane-receiver complexesmay be generated through fusion of erythrocytes, e.g., betweenerythrocytes autologous to the subject and one or more allogeneicerythrocytes, liposomes, and/or artificial vesicles.

In certain embodiments, autologous transfusion of syntheticmembrane-receiver complexes includes isolating erythrocytes,reticulocytes or hematopoietic stem cells from a subject, generating asuitable synthetic membrane-receiver complex by contacting the cell witha receiver by methods described herein and administering (e.g., bytransfusion) the synthetic membrane-receiver complex into the samesubject.

In certain embodiments, allogeneic transfusion of syntheticmembrane-receiver complexes includes isolating erythrocytes,reticulocytes or hematopoietic stem cells from a donor, generating asuitable synthetic membrane-receiver complex by contacting the cell witha receiver by methods described herein and administering (e.g., bytransfusion) the synthetic membrane-receiver complex into a subject thatis different from the donor. Where allogeneic cells are used fortransfusion, care needs to be taken to use a compatible ABO blood groupto prevent an acute intravascular hemolytic transfusion reaction whichis characterized by complement activation and lysis of incompatibleerythrocytes. The ABO blood types are defined based on the presence orabsence of the blood type antigens A and B, monosaccharide carbohydratestructures that are found at the termini of oligosaccharide chainsassociated with glycoproteins and glycolipids on the surface of theerythrocytes (reviewed in Liu et al., Nat. Biotech. 25:454-464 (2007)).Group 0 erythrocytes lack either of these antigenic monosaccharidestructures. Subjects with group A erythrocytes have naturally occurringantibodies to group B erythrocytes whereas subjects with group Berythrocytes have antibodies to group A erythrocytes. Blood group ABsubjects have neither antibody and blood group 0 individuals have both.Subjects with either anti-A and/or anti-B antibodies cannot receive atransfusion of blood containing the corresponding antigen. Because group0 erythrocytes contain neither A nor B antigens, they can be safelytransfused into recipients of any ABO blood group, e.g., group A, B, AB,or O recipients. Group O erythrocytes are considered universal and maybe used in all blood transfusions. In contrast, group A erythrocytes maybe given to group A and AB recipients, group B erythrocytes may be givento group B and AB recipients, and group AB erythrocytes may only begiven to AB recipients. In embodiments in which syntheticmembrane-receiver complexes are generated by contecting erythrocytes ortheir precursors with a receiver the sourced erythrocytes or theirprecursors are matched for compatibility with the recipient.

In some instances, it may be beneficial to convert a syntheticmembrane-receiver complex comprising a non-group 0 erythrocyte to auniversal blood type. Enzymatic removal of the immunodominantmonosaccharides on the surface of group A and group B erythrocytes maybe used to generate a population of group O-like syntheticmembrane-receiver complexes (See, e.g., Liu et al., Nat. Biotech.25:454-464 (2007)). Group B synthetic membrane-receiver complexes may beconverted using an α-galactosidase derived from green coffee beans.Alternatively or in addition, α-N-acetylgalactosaminidase andα-galactosidase enzymatic activities derived from E. meningosepticumbacteria may be used to respectively remove the immunodominant A and Bantigens (Liu et al., Nat. Biotech. 25:454-464 (2007)), if present onthe synthetic membrane-receiver complexes. In one example, packed redblood cells isolated as described herein, are incubated in 200 mMglycine (pH 6.8) and 3 mM NaCl in the presence of eitherα-N-acetylgalactosaminidase and α-galactosidase (about 300 μg/ml packedred blood cells) for 60 min at 26° C. After treatment, the red bloodcells are washed by 3-4 rinses in saline with centrifugation andABO-typed according to standard blood banking techniques.

In specific embodiments, the synthetic membrane-receiver complexesdescribed herein may be generated in the following way. First, erythroidprecursor cells are isolated. These cells may alternatively beautologous to the patient or from substantially universal donor blood.For example, the cells may be ABO type O, rhesus factor Rh r/r, Duffy−/−, and large Kell antigen K1 negative. In the course ofdifferentiation from erythroid precursor cell to erythroid cell, anexogenous nucleic acid encoding the receiver is introduced. theexogenous nucleic acid encoding the receiver can be under the control ofan erythroid-specific promoter, such as a GATA-1 promoter (see e.g.,Repik et al., Clin Exp Immunol 2005, 140:230). the exogenous nucleicacid encoding the receiver can be introduced in any way known in theart, for example, as plasmid DNA, virus, or mRNA. Nucleic acidintroduction can be achieved by a variety of standard methods, e.g.,transfection, transduction, or electroporation.

In specific embodiments, the synthetic membrane-receiver complexesdescribed herein may be generated by contacting platelets with areceiver. Each day an adult human produces 2×10¹¹ red blood cells, andabout one-half as many white cells and platelets. In humans, nearly allblood cell production occurs in the red bone marrow that represents ahierarchical developmental system composed of hematopoietic stem cells,intermediate level progenitors and maturing cells committed to eachlineage.

Although the morphology of all the major blood cell types is similarthrough their initial development stages, megakaryocytes, cellscommitted to platelet production, are marked by an obvious structuraland functional departure beyond the blast cell level of differentiationgrowing to a size 10 times the diameter of most other bone marrow andblood cells, and containing up to 128 times the normal chromosomalcomplement, these cells give rise to blood platelets. After a series ofnormal cell divisions, the developing megakaryocyte precursor enters aunique cell cycle characterized by a brief (about 1 h) G1 phase, atypical (7 h) S phase, a very brief (^(˜)45 min) G2 phase, followed bythe endomitotic phase (an aborted M phase). Once the cell develops ahighly polyploid nucleus, it also develops demarcation membranesnecessary for cytoplasmic fragmentation. This event is accompanied byexpression of glycoprotein GPIIbIIIa (platelet fibrinogen receptor;Papayannopoulou et al., Exp. Hematol., 24: 660-9, 1996) and GPIb (vonWillibrand factor receptor; Kaushansky et al., Nature, 369: 568-571,1994), the granules that contain ADP, serotonin, -thromboglobulin, andother substances critical for mature platelet function. Finally, highlypolyploid megakaryocytes undergo cytoplasmic partitioning, allowing therelease of thousands of platelets (Choi et al., Blood, 85: 402-413,1995; Cramer et al., Blood, 89: 2336-2346, 1997).

Like all blood cell precursors, megakaryocytes are derived frompluripotent marrow stem cellsthat retain the capacity to extensivelyself-renew, or to differentiate into all of the elements of the blood.Platelet production is in part regulated by signaling mechanisms inducedby interaction between thrombopoietin (TPO) and its cellular receptorTPOR/MPUc-MPL.

Thrombopoietin (TPO) is a hematopoietic growth factor involved instimulation of megakaryocytopoiesis and platelet production. TPO isexpressed in liver and kidney, and, in response to platelet demand, itsexpression may be also upregulated in the bone marrow microenvironment(Kato et al., Stem Cells, 16: 322-328, 1998; McCarty et al., Blood,86:3668-3675, 1995). As TPO expression is mostly constitutive, the TPOlevels are believed to be regulated by sequestering by platelets(Fielder et al., Blood 87: 2154, 1996).

The gene encoding TPO has been cloned and characterized (Kuter et al.,Proc. Natl. Acad. Sci. USA, 91:11104-11108, 1994; Bartley et al., Cell,77:1117-1124, 1994; Kaushansky et al., Nature, 369:568-571, 1994;Wendling et al., Nature, 369:571-574, 1994, and de Sauvage et al.,Nature, 369:533-538, 1994). Human TPO (hTPO) cDNA encodes a 353 aminoacid-long polypeptide. The full-length hTPO secreted from mammaliancells after cleavage of the signal peptide consists of 332 amino acids.Although the predicted molecular mass of this protein is 38 kD, themolecular masses reported from measurements of material in serum or inculture fluid from recombinant cells vary from 18 to 85 kD(glycosylation, and post-translational proteolytic processing).

The cell surface receptor for TPO (TPOR/MPL/c-MPL) is a product of theprotooncogene c-mpl, a homologue of v-mpl, an envelope protein of themyeloproliferative leukaemia virus (MPLV) shown to induce a pan-myeloiddisorder (Wendling, Virol., 149:242-246, 1986). The human c-mpl genecodes for a protein of 635 aa having a predicted molecular weight of 71kD (Vigon et al., Proc. Natl. Acad. Sci. USA, 89:5640-44, 1992; Mignotteet al., Genomics, 20: 5-12, 1994).

Mice rendered null for the expression of either TPO or its receptor(TPOR/MPL/c-MPL) manifest a severe thrombocytopenic phenotype (Gurney etal., Science, 265: 1445, 1994; Kaushansky et al., J. Clin. Invest., 96:1683, 1995; de Sauvage et al., J. Exp. Med., 183: 651, 1996).

Multiple cytokines (e.g., stem cell factor [SCF], IL-1, IL-3, IL-6,IL-11, leukaemia inhibiting factor [LIF], G-CSF, GM-CSF, M-CSF,erythropoietin (EPO), kit ligand, and -interferon) have been shown topossess thrombocytopoietic activity.

The resulting platelets are small disc-shaped cell fragments whichundergo a rapid transformation when they encounter sites of vasculardamage. They become more spherical and extrude pseudopodia, theirfibrinogen receptors are activated leading to aggregation, and theyrelease their granule contents and eventually they form a plug which isresponsible for primary hemostasis (Siess, W., Physiol. Rev. 69: 58-178,1989). Activation of platelets is also implicated in the pathogenesis ofunstable angina, myocardial infarction and stroke (Packham, M. A., CanJ. Physiol Pharmacol. 72: 278-284).

Several physiological substances are involved in the activation ofplatelets such as collagen, which is exposed at the subendothelialsurfaces, thrombin, generated by the coagulation cascade, andthromboxane A2 (TXA₂) and ADP, which are released from activatedplatelets. Collagen binds to several platelet membrane proteinsincluding integrin α2β1 leading to platelet activation through therelease of TXA₂ and ADP (Shattil, S. J., et al., Curr. Opin. Cell Biol.6: 695-704, 1994). In contrast, thrombin, TXA₂, and ADP, activateG-protein coupled receptors directly and induce platelet aggregation andgranule release (Hourani, S. M, and Cusack, N. J., Pharmacol. Rev. 43:243-298, 1991). The major events involved in platelet activation arebelieved to be the result of the activation of β-isoforms ofphospholipase C (PLC) leading to the generation of inositol 1,4,5triphosphate and diacylglycerol. Platelets mainly contain two isoforms,PLC-β2 and PLC-β3.

Platelet receptors which mediate platelet adhesion and aggregation arelocated on the two major platelet surface glycoprotein complexes. Thesecomplexes are the glycoprotein Ib-IX complex which facilitates plateletadhesion by binding von Willebrand factor (vWF), and the glycoproteinIIb-IIIa complex which links platelets into aggregates by binding tofibrinogen. Patients with the Bernard-Soulier syndrome, a congenitalbleeding disorder, show deficient platelet adhesion due to a deficiencyin the glycoprotein Ib-IX complex which binds vWF, mildthrombocytopenia, and large lymphocoid platelets.

Glycoprotein V (GPV) is a major (≈12,000 molecules/platelet), heavilyglycosylated platelet membrane protein (Mr 82,000). Exposure ofplatelets to thrombin liberates a 69 kDa soluble fragment termed GPVfl.GPV can interact non-covalently with the GPIb-IX complex a complexformed by the non-covalent association of GPIb (consisting of GPIbα, a145 kDa protein, disulfide linked to GPIbβ, a 24 kDa protein) with GPIX(a 22 kDa protein). The binding sites for von Willebrand factor and forthrombin on the GPIb-IX complex have been localized on GPIbα. Sincethrombin is now known to activate platelets by cleaving the thrombinreceptor (Vu et. al., Cell 64:1057-1068 (1990)), a G-protein coupledreceptor, it is unknown whether thrombin cleaves GPV incidently as aconsequence of thrombin binding to GPIbα, or whether this cleavage has aphysiological role. GPIBα, GPIBβ, and GPIX contain one or morehomologous 24 amino acid leucine-rich domains. These domains are alsofound in a large family of leucine-rich glycoproteins (LRG).

GPV is a marker for the megakaryocytic cell lineage. A monoclonalantibody specific for GPV (SW16) does not bind to red cells, leukocyteseendothelial cells, or cell lines such as HEL or MEG-01 which are knownto express platelet megakaryocyte markers.

Mature GPV is composed of 543 amino acids which contain a singletransmembrane domain, a short cytoplasmic domain (16 residues) and alarge extracellular domain with 8 potential N-glycosylation sites.Analysis of the extracellular domain revealed the presence of 15 tandemLeu-rich repeats of 24 amino acids with homology to GPIba, andidentified a cleavage site for thrombin near the C-terminus withhomology to the Act chain of fibrinogen.

Culturing

Sources for generating synthetic membrane-receiver complexes describedherein include circulating cells such as erythroid cells. A suitablecell source may be isolated from a subject as described herein frompatient-derived hematopoietic or erythroid progenitor cells, derivedfrom immortalized erythroid cell lines, or derived from inducedpluripotent stem cells, optionally cultured and differentiated. Methodsfor generating erythrocytes using cell culture techniques are well knownin the art, e.g., Giarratana et al., Blood 2011, 118:5071, Huang et al.,Mol Ther 2013, epub ahead of print September 3, or Kurita et al., PLOSOne 2013, 8:e59890. Protocols vary according to growth factors, startingcell lines, culture period, and morphological traits by which theresulting cells are characterized. Culture systems have also beenestablished for blood production that may substitute for donortransfusions (Fibach et al. 1989 Blood 73:100). Recently, CD34+ cellswere differentiated to the reticulocyte stage, followed by successfultransfusion into a human subject (Giarratana et al., Blood 2011,118:5071).

Provided herein are culturing methods for erythroid cells and syntheticmembrane-receiver complexes derived from erythroid cells. Erythroidcells can be cultured from hematopoietic progenitor cells, including,for example, CD34+ hematopoietic progenitor cells (Giarratana et al.,Blood 2011, 118:5071), induced pluripotent stem cells (Kurita et al.,PLOS One 2013, 8:e59890), and embryonic stem cells (Hirose et al. 2013Stem Cell Reports 1:499). Cocktails of growth and differentiationfactors that are suitable to expand and differentiate progenitor cellsare known in the art. Examples of suitable expansion and differentiationfactors include, but are not limited to, stem cell factor (SCF), aninterleukin (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-11, IL-12, CSF, G-CSF, thrombopoietin (TPO), GM-CSF,erythropoietin (EPO), Flt3, Flt2, PIXY 321, and leukemia inhibitoryfactor (LIF).

Erythroid cells can be cultured from hematopoietic progenitors, such asCD34+ cells, by contacting the progenitor cells with defined factors ina multi-step culture process. For example, erythroid cells can becultured from hematopoietic progenitors in a three-step process.

The first step may comprise contacting the cells in culture with stemcell factor (SCF) at 1-1000 ng/mL, erythropoietin (EPO) at 1-100 U/mL,and interleukin-3 (IL-3) at 0.1-100 ng/mL. The first step optionallycomprises contacting the cells in culture with a ligand that binds andactivates a nuclear hormone receptor, such as e.g., the glucocorticoidreceptor, the estrogen receptor, the progesterone receptor, the androgenreceptor, or the pregnane x receptor. The ligands for these receptorsinclude, for example, a corticosteroid, such as, e.g., dexamethasone at10 nM-100 μM or hydrocortisone at 10 nM-100 μM; an estrogen, such as,e.g., beta-estradiol at 10 nM-100 μM; a progestogen, such as, e.g.,progesterone at 10 nM-100 μM, hydroxyprogesterone at 10 nM-100 μM,5a-dihydroprogesterone at 10 nM-100 μM, 11-deoxycorticosterone at 10nM-100 μM, or a synthetic progestin, such as, e.g., chlormadinoneacetate at 10 nM-100 μM; an androgen, such as, e.g., testosterone at 10nM-100 μM, dihydrotestosterone at 10 nM-100 μM or androstenedione at 10nM-100 μM; or a pregnane x receptor ligand, such as, e.g., rifampicin at10 nM-100 hyperforin at 10 nM-100 μM, St. John's Wort (hypericin) at 10nM-100 μM, or vitamin E-like molecules, such as, e.g., tocopherol at 10nM-100 μM. The first step may also optionally comprise contacting thecells in culture with an insulin-like molecule, such as, e.g., insulinat 1-50 μg/mL, insulin-like growth factor 1 (IGF-1) at 1-50 μg/mL,insulin-like growth factor 2 (IGF-2) at 1-50 μg/mL, or mechano-growthfactor at 1-50 μg/mL. The first step further may optionally comprisecontacting the cells in culture with transferrin at 0.1-5 mg/mL.

The first step may optionally comprise contacting the cells in culturewith one or more interleukins (IL) or growth factors such as, e.g.,IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,granulocyte colony-stimulating factor (G-CSF), macrophagecolony-stimulating factor (M-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), thrombopoietin, fibroblast growthfactor (FGF), platelet-derived growth factor (PDGF), transforming growthfactor beta (TGF-B), tumor necrosis factor alpha (TNF-A), megakaryocytegrowth and development factor (MGDF), leukemia inhibitory factor (LIF),and Flt3 ligand. Each interleukin or growth factor may typically besupplied at a concentration of 0.1-100 ng/mL. The first step may alsooptionally comprise contacting the cells in culture with serum proteinsor non-protein molecules such as, e.g., fetal bovine serum (1-20%),human plasma (1-20%), plasmanate (1-20%), human serum (1-20%), albumin(0.1-100 mg/mL), or heparin (0.1-10 U/mL).

The second step may comprise contacting the cells in culture with stemcell factor (SCF) at 1-1000 ng/mL and erythropoietin (EPO) at 1-100U/mL. The second step may also optionally comprise contacting the cellsin culture with an insulin-like molecule, such as e.g., insulin at 1-50μg/mL, insulin-like growth factor 1 (IGF-1) at 1-50 μg/mL, insulin-likegrowth factor 2 (IGF-2) at 1-50 μg/mL, or mechano-growth factor at 1-50μg/mL. The second step may further optionally comprise contacting thecells in culture with transferrin at 0.1-5 mg/mL. The second may alsooptionally comprise contacting the cells in culture with serum proteinsor non-protein molecules such as, e.g., fetal bovine serum (1-20%),human plasma (1-20%), plasmanate (1-20%), human serum (1-20%), albumin(0.1-100 mg/mL), or heparin (0.1-10 U/mL).

The third step may comprise contacting the cells in culture witherythropoietin (EPO) at 1-100 U/mL. The third step may optionallycomprise contacting the cells in culture with stem cell factor (SCF) at1-1000 ng/mL. The third step may further optionally comprise contactingthe cells in culture with an insulin-like molecule, such as e.g.,insulin at 1-50 μg/mL, insulin-like growth factor 1 (IGF-1) at 1-50μg/mL, insulin-like growth factor 2 (IGF-2) at 1-50 μg/mL, ormechano-growth factor at 1-50 μg/mL. The third step may also optionallycomprise contacting the cells in culture with transferrin at 0.1-5mg/mL. The third step may also optionally comprise contacting the cellsin culture with serum proteins or non-protein molecules such as, e.g.,fetal bovine serum (1-20%), human plasma (1-20%), plasmanate (1-20%),human serum (1-20%), albumin (0.1-100 mg/mL), or heparin (0.1-10 U/mL).

In some embodiments, methods of expansion and differentiation of thesynthetic membrane-receiver complexes do not include culturing thesynthetic membrane-receiver complexes in a medium comprising amyeloproliferative receptor (mpl) ligand.

The culture process may optionally comprise contacting cells by a methodknown in the art with a molecule, e.g., a DNA molecule, an RNA molecule,a mRNA, an siRNA, a microRNA, a lncRNA, a shRNA, a hormone, or a smallmolecule, that activates or knocks down one or more genes. Target genescan include, for example, genes that encode a transcription factor, agrowth factor, or a growth factor receptor, including but not limitedto, e.g., GATA1, GATA2, CMyc, hTERT, p53, EPO, SCF, insulin, EPO-R,SCF-R, transferrin-R, insulin-R.

In one embodiment, CD34+ cells are placed in a culture containingvarying amounts of IMDM, FBS, glutamine, BSA, holotransferrin, insulin,dexamethasone, β-estradiol, IL-3, SCF, and erythropoietin, in threeseparate differentiation stages for a total of 22 days.

In one embodiment, CD34+ cells are placed in a culture containingvarying amounts of IMDM, FBS, glutamine, BSA, holotransferrin, insulin,dexamethasone, β-estradiol, IL-3, SCF, and thrombopoietin, in threeseparate differentiation stages for a total of 14 days.

In one embodiment, CD34+ cells are placed in a culture containingvarying amounts of IMDM, FBS, glutamine, BSA, holotransferrin, insulin,dexamethasone, β-estradiol, IL-3, SCF, and GCSF, in three separatedifferentiation stages for a total of 15 days.

Compositions

Provided herein are pharmaceutical compositions comprising syntheticmembrane-receiver complexes that are suitable for administration to asubject. The pharmaceutical compositions generally comprise a populationof synthetic membrane-receiver complexes and apharmaceutically-acceptable carrier in a form suitable foradministration to a subject. Pharmaceutically-acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations ofpharmaceutical compositions comprising a population of syntheticmembrane-receiver complexes. (See, e.g., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). Thepharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Pharmaceutically-acceptable excipients include excipients that aregenerally safe, non-toxic, and desirable, including excipients that areacceptable for veterinary use as well as for human pharmaceutical use.Such excipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

Examples of carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and compounds for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or compound is incompatible with the synthetic membrane-receivercomplexes described herein, use thereof in the compositions iscontemplated. Supplementary therapeutic agents may also be incorporatedinto the compositions. Typically, a pharmaceutical composition isformulated to be compatible with its intended route of administration.The synthetic membrane-receiver complexes can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intradermal, transdermal, rectal, intracranial, intraperitoneal,intranasal; intramuscular route or as inhalants. The syntheticmembrane-receiver complexes can optionally be administered incombination with other therapeutic agents that are at least partlyeffective in treating the disease, disorder or condition for which thesynthetic membrane-receiver complexes are intended.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates, and compounds for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, e.g., water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, e.g., by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalcompounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic compounds, e.g., sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition a compound which delays absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thesynthetic membrane-receiver complexes in an effective amount and in anappropriate solvent with one or a combination of ingredients enumeratedherein, as desired. Generally, dispersions are prepared by incorporatingthe synthetic membrane-receiver complexes into a sterile vehicle thatcontains a basic dispersion medium and any desired other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The synthetic membrane-receiver complexes can be administered in theform of a depot injection or implant preparation which can be formulatedin such a manner to permit a sustained or pulsatile release of thesynthetic membrane-receiver complexes, their receiver(s) and/or theiroprional payload(s).

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, thesynthetic membrane-receiver complexes can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding compounds, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegratingcompound such as alginic acid, Primogel, or corn starch; a lubricantsuch as magnesium stearate or Sterotes; a glidant such as colloidalsilicon dioxide; a sweetening compound such as sucrose or saccharin; ora flavoring compound such as peppermint, methyl salicylate, or orangeflavoring.

For administration by inhalation, the synthetic membrane-receivercomplexes are delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer.

Systemic administration of compositions comprising syntheticmembrane-receiver complexes can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, e.g., fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the modified red blood cells are formulated into ointments, salves,gels, or creams as generally known in the art.

The synthetic membrane-receiver complexes can also be prepared aspharmaceutical compositions in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In some embodiments, the synthetic membrane-receiver complexes areprepared with carriers that will decrease the rate with which syntheticmembrane-receiver complexes are eliminated from the body of a subject.For example, controlled release formulation are suitable, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.

In one embodiment the pharmaceutical composition comprising syntheticmembrane-receiver polypeptide complexes is administered intravenouslyinto a subject that would benefit from the pharmaceutical composition.In other embodiments, the composition is administered to the lymphaticsystem, e.g., by intralymphatic injection or by intranodal injection(see e.g., Senti et al., 2008 PNAS 105(46):17908), or by intramuscularinjection, by subcutaneous administration, by direct injection into thethymus, or into the liver.

In one embodiment, the pharmaceutical composition comprising syntheticmembrane-receiver polypeptide complexes is administered as a liquidsuspension. In one embodiment the pharmaceutical composition isadministered as a coagulated formulation that is capable of forming adepot following administration, and in a preferred embodiment slowlyrelease synthetic membrane-receiver polypeptide complexes intocirculation, or in a preferred embodiment remain in depot form.

In one embodiment, the pharmaceutical composition comprising syntheticmembrane-receiver complexes is stored using methods and buffercompositions that are capable of maintaining viability of the syntheticmembrane-receiver complexes. For example, deoxygenation prior to storageto maintain an anaerobic state, manipulation of pH, supplementation ofmetabolic precursors, manipulation of osmotic balance, increasing of thevolume of the suspending medium, and/or reduction of oxidative stress byadding protective molecules can be used to maintain the viability of thesynthetic membrane-receiver complexes. Several studies employing acombination of these strategies have reported maintenance of viabilityof erythrocytes allowing an extension of storage beyond 6 weeks (seee.g., Yoshida and Shevkoplyas, Blood Transfus 2010 8:220).

Pharmaceutically acceptable carriers or excipients may be used todeliver the synthetic membrane-receiver polypeptides described herein.Excipient refers to an inert substance used as a diluent or vehicle.Pharmaceutically acceptable carriers are used, in general, with acompound so as to make the compound useful for a therapy or as aproduct. In general, for any substance, a pharmaceutically acceptablecarrier is a material that is combined with the substance for deliveryto a subject. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable. Insome cases the carrier is essential for delivery, e.g., to solubilize aninsoluble compound for liquid delivery; a buffer for control of the pHof the substance to preserve its activity; or a diluent to prevent lossof the substance in the storage vessel. In other cases, however, thecarrier is for convenience, e.g., a liquid for more convenientadministration. Pharmaceutically acceptable salts of the compoundsdescribed herein may be synthesized according to methods known to thoseskilled in the arts.

Typically, pharmaceutically acceptable compositions are highly purifiedto be free of contaminants, are biocompatible and not toxic, and aresuited to administration to a subject. If water is a constituent of thecarrier, the water is highly purified and processed to be free ofcontaminants, e.g., endotoxins.

The pharmaceutically acceptable carrier may be lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate,alginates, gelatin, calcium silicate, micro-crystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and/or mineral oil, but is not limited thereto. Thepharmaceutical composition may further include a lubricant, a wettingagent, a sweetener, a flavor enhancer, an emulsifying agent, asuspension agent, and/or a preservative.

Provided are pharmaceutical compositions containing syntheticmembrane-receiver complexes having effective levels of receivers. Suchcompositions contain a plurality of synthetic membrane-receivercomplexes, e.g., 1×10³ complexes, or 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or greater than 1×10¹² complexes. Inspecific examples, synthetic membrane-receiver complexes generated fromerythroid cells may be administered as packed red blood cells in asaline solution at a concentration of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or greater than 90% mass to volume ratio (% m/v). The time ofadministration to a patient may range from 10 minutes to four hours, ormore.

In specific examples, synthetic membrane-receiver complexes generatedfrom erythroid cells can be stored in an appropriate buffer, e.g., anFDA-approved anticoagulant preservative solution such as anticoagulantcitrate-dextrose A (ACD-A), citrate-phosphate dextrose (CPD),Citratephosphate-dextrose-dextrose (CP2D), orcitrate-phosphate-dextrose-adenine (CPDA-1). The compositions may bestored for up to 21 days.

Alternatively, synthetic membrane-receiver complexes generated fromerythroid cells can be stored in an approved additive solution, e.g.,AS-1 (Adsol), AS-3 (Nutricel), AS-5 (Optisol), or AS-7 (SOLX).

Alternatively, synthetic membrane-receiver complexes generated fromerythroid cells can stored in a glycerol cryoprotective solution. Thecompositions may be frozen and stored for up to 10 years. Frozen cellsmay be thawed and deglycerolized by successive washing steps, forexample with 0.9% sodium chloride before use.

Provided herein are compositions and pharmaceutical compositionscomprising a plurality of cultured functional erythroid cells thatcomprise a receiver. The compositions and pharmaceutical compositionsmay comprise a solution of appropriate storage buffer such as, e.g.,anticoagulant citrate-dextrose A. The compositions and pharmaceuticalcompositions comprising the plurality of cultured functional erythroidcells that comprise a receiver may additionally comprise an approvedadditive such as, e.g., Adsol. The compositions and pharmaceuticalcompositions comprising the plurality of cultured functional erythroidcells that comprise receiver may additionally comprise a glycerolcryoprotective solution for frozen storage.

In one embodiment, the synthetic membrane-receiver polypeptide complexis able to form a multi-complex aggregate, e.g., a dimer, a trimer, amultimer, with another synthetic membrane-receiver polypeptide complex.

In one embodiment the synthetic membrane-receiver polypeptide complex isable to form a multi-complex aggregate, e.g., a dimer, a trimer, amultimer, with component of the circulatory system, e.g an erythrocyte,a reticulocyte, a platelet, a macrophage, a lymphocyte, a T cell, a Bcell, a mast cell.

The dosing and frequency of the administration of the syntheticmembrane-receiver complexes and pharmaceutical compositions thereof canbe determined by the attending physician based on various factors suchas the severity of disease, the patient's age, sex and diet, theseverity of any inflammation, time of administration, and other clinicalfactors. In one example, an intravenous administration is initiated at adose which is minimally effective, and the dose is increased over apre-selected time course until a positive effect is observed.Subsequently, incremental increases in dosage are made limiting tolevels that produce a corresponding increase in effect while taking intoaccount any adverse affects that may appear.

Non-limited examples of suitable dosages can range, for example, from1×10¹⁰ to 1×10¹⁴, from 1×10¹¹ to 1×10¹³, or from 5×10¹¹ to 5×10¹²synthetic membrane-receiver complexes. Specific examples include about5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², or more syntheticmembrane-receiver complexes. Each dose of synthetic membrane-receivercomplexes can be administered at intervals such as once daily, onceweekly, twice weekly, once monthly, or twice monthly.

“Complex-based proportional dosage” is the number of syntheticmembrane-receiver complexes administered as a dose relative to anaturally occurring quantity of circulating entities. The circulatingentities may be cells, e.g., erythrocytes, reticulocytes, orlymphocytes, or targets, e.g., antigens, antibodies, viruses, toxins,cytokines, etc. The units are defined as synthetic membrane-receivercomplex per circulating entity, ie SCMRC/CE. This dosage unit mayinclude 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, 1, 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹.

The pharmaceutical compositions described herein comprise a syntheticmembrane-receiver complex and optionally a pharmaceutically active ortherapeutic agent. The therapeutic agent can be a biological agent, asmall molecule agent, or a nucleic acid agent.

Dosage forms are provided that comprise a pharmaceutical compositioncomprising a synthetic membrane-receiver complex described herein. Insome embodiments, the dosage form is formulated as a liquid suspensionfor intravenous injection.

Medical devices are provided that comprise a container holding apharmaceutical composition comprising a synthetic membrane-receivercomplex described herein and an applicator for intravenous injection ofthe pharmaceutical composition to a subject.

Medical kits are provided that comprise a pharmaceutical compositioncomprising a synthetic membrane-receiver complex described herein and amedical device for intravenous injection of the pharmaceuticalcomposition to a subject.

A pharmaceutically acceptable suspension of synthetic membrane-receivercomplexes is preferably packaged in a volume of approximately 10 toapproximately 250 ml. The packaging can be a syringe or an IV bagsuitable for transfusions. Administration of the suspension is carriedout, e.g., by intravenous or intra-arterial injection, optionally usinga drip from an IV bag or the like. The administration is typicallycarried out intravenously in the arm or via a central catheter. Foradministrations exceeding 50 ml use of a drip is preferred.

Processes and Properties

In some embodiments, the membrane-receiver complex is generated using aprecursor hematopoietic cell, e.g., a CD34+ cell, an erythrocyte, aplatelet, a megakaryocyte, or a neutrophil as a source. In someembodiments, the precursor hematopoietic cell is isolated from a humandonor by a GMP-compliant process. In some embodiments, the startingcells are sourced from an autologous donor. In some embodiments, thestarting cells are sourced from an allogeneic donor. The donor may betyped for blood cell antigen polymorphisms and/or the donor is genotypedfor blood cell antigens. The donor can be a universal blood donor. Insome embodiments, the donor has the Bombay phenotype, ie. does notexpress the H antigen. In some embodiments, the donor has ABO blood typeO and is Rh-negative.

In some embodiments, the membrane-receiver complex is generated usingCD34+ hematopoietic progenitor cells, mobilized peripheral CD34+ cells,or bone marrow-derived CD34+ cells as a source for the startingmaterial. In some embodiments, the starting cells are derived fromumbilical cord blood, are induced pluripotent stem cells or areembryonic stem cells.

The synthetic membrane-receiver complex may be cultured. Culturedcomplexes can be scaled up from bench-top scale to bioreactor scale. Forexample, the complexes are cultured until they reach saturation density,e.g., 1×10⁵, 1×10⁶, 1×10⁷, or greater than 1×10⁷ complexes per ml.Optionally, upon reaching saturation density, the complexes can betransferred to a larger volume of fresh medium. The membrane-receivercomplexes may be cultured in a bioreactor, such as, e.g., a Wave-typebioreactor, a stirred-tank bioreactor. Various configurations ofbioreactors are known in the art and a suitable configuration may bechosen as desired. Configurations suitable for culturing and/orexpanding populations of synthetic membrane-receiver complexes caneasily be determined by one of skill in the art without undueexperimentation. The bioreactor can be oxygenated. The bioreactor mayoptionally contain one or more impellers, a recycle stream, a mediainlet stream, and control components to regulate the influx of media andnutrients or to regulate the outflux of media, nutrients, and wasteproducts.

In some embodiments, the bioreactor may contain a population of humanfunctional erythroid cells comprising a receiver that shed theirintracellular DNA over the course of the culture process. For example,the bioreactor may contain a population of human erythroid cells,enucleated erythroid cells, and pyrenocytes after culture. In a specificembodiment, the human erythroid cells and enucleated erythroid cellscomprise a receiver and the receiver is retained by the enucleatederythroid cell, whereas the exogenous nucleic acid encoding the receiveris not retained by the enucleated cell. In certain embodiments, theenucleated functional erythroid cell comprising the receiver exhibitssubstantially the same osmotic membrane fragility as a correspondingisolated unmodified, uncultured erythroid cell.

In one embodiment. The population of synthetic membrane-receivercomplexes generated from erythroid cells or erythroid cell precursors inthe bioreactor undergo a total expansion of greater than 20,000-fold in14 days or greater. In some embodiments, the receiver is introduced intoa cultured or freshly isolated erythroid cell precursor and afterintroduction of an exogenous nucleic acid encoding the receiver thepopulation of synthetic membrane-receiver complexes generated from theerythroid cell precursors in the bioreactor expands in the bioreactorfrom the precursor cells by more than 20,000-fold.

In some embodiments, the bioreactor is a Wave bioreactor or aimpeller-driven agitator. The bioreactor may be aerated by means of asparger. In one embodiment, the bioreactor is disposable. In oneembodiment, the bioreactor is CIP (cleaned in place). The finalcomplexes number of synthetic membrane-receiver complexes that may beobtained in a bioreactor setting as described herein can be greater than10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or greater than 10¹³ complexes. The densityof synthetic membrane-receiver complexes may be monitored during cultureby measuring cell density by hemacytometer counting or by opticaldensity reading at 600 nm. Optionally, the culture process is monitoredfor pH levels, oxygenation, agitation rate, and/or recycle rate.

The identity of the membrane-receiver complexes can be assessed by invitro assays. For example, the identity of the membrane-receivercomplexes is assessed by counting the number of complexes in apopulation, e.g., by microscopy, by flow cytometry, or by hemacytometry.Alternatively or in addition, the identity of the membrane-receivercomplexes is assessed by analysis of protein content of the complex,e.g., by flow cytometry, Western blot, immunoprecipitation, fluorescencespectroscopy, chemiluminescence, mass spectrometry, or absorbancespectroscopy. In one embodiment, the protein content assayed is anon-surface protein, e.g., an integral membrane protein, hemoglobin,adult hemoglobin, fetal hemoglobin, embryonic hemoglobin, a cytoskeletalprotein. In one embodiment, the protein content assayed is a surfaceprotein, e.g., a differentiation marker, a receptor, a coreceptor, atransporter, a glycoprotein. In one embodiment, the surface protein isselected from the list including, but not limited to, glycophorin A,CKIT, transferrin receptor, Band3, Kell, CD45, CD46, CD47, CD55, CD59,CR1. In some embodiments, the identity of the membrane-receivercomplexes is assessed by analysis of the receiver content of thecomplex, e.g., by flow cytometry, Western blot, immunoprecipitation,fluorescence spectroscopy, chemiluminescence, mass spectrometry, orabsorbance spectroscopy. For example, the identity of themembrane-receiver complexes can be assessed by the mRNA content of thecomplexes, e.g., by RT-PCR, flow cytometry, or northern blot. Theidentity of the membrane-receiver complexes can be assessed by nuclearmaterial content, e.g., by flow cytometry, microscopy, or southern blot,using, e.g., a nuclear stain or a nucleic acid probe. Alternatively orin addition, the identity of the membrane-receiver complexes is assessedby lipid content of the complexes, e.g by flow cytometry, liquidchromatography, or by mass spectrometry.

In some embodiments, the identity of the membrane-receiver complexes isassessed by metabolic activity of the complexes, e.g by massspectrometry, chemiluminescence, fluorescence spectroscopy, absorbancespectroscopy. Metabolic activity can be assessed by ATP consumption rateand/or the metabolic activity is assessed measuring2,3-diphosphoglycerate (2,3-DPG) level in the syntheticmembrane-receiver complex. The metabolic activity can be assessed as therate of metabolism of one of the following, including but not limitedto, Acetylsalicylic acid, N-Acetylcystein, 4-Aminophenol, Azathioprine,Bunolol, Captopril, Chlorpromazine, Dapsone, Daunorubicin,Dehydroepiandrosterone, Didanosin, Dopamine, Epinephrine, Esmolol,Estradiol, Estrone, Etoposide, Haloperidol, Heroin, Insulin,Isoproterenol, Isosorbide dinitrate, LY 217896, 6-mercaptopurine,Misonidazole, Nitroglycerin, Norepinephrine, Para-aminobenzoic acid. Insome embodiments, the identity of the membrane-receiver complexes isassessed by partitioning of a substrate by the complexes, e.g by massspectrometry, chemiluminescence, fluorescence spectroscopy, orabsorbance spectroscopy. The substrate can be one of the following,including but not limited to, Acetazolamide, Arbutine, Bumetamide,Creatinine, Darstine, Desethyldorzolamide, Digoxigenin digitoxoside,Digoxin-16′-glucuronide, Epinephrine, Gentamycin, Hippuric acid,Metformin, Norepinephrine, p-Aminohippuric acid, Papaverine, PenicillinG, Phenol red, Serotonin, Sulfosalicylic acid, Tacrolimus, Tetracycline,Tucaresol, and Vancomycin.

In one embodiment, the population of synthetic membrane-receivercomplexes is differentiated from a precursor cell or complex. In thisembodiment, the differentiation state of the population of syntheticmembrane-receiver complexes is assessed by an in vitro assay. The invitro assays include those described herein for assessing the identityof the complexes, including but not limited to expansion rate, number,protein content or expression level, mRNA content or expression level,lipid content, partition of a substrate, catalytic activity, ormetabolic activity.

In some embodiments, the membrane-receiver complexes are cultured andthe differentiation state of the complexes is assessed at multiple timepoints over the course of the culture process.

Synthetic membrane-receiver complexes may be generated usingreticulocytes as a source for starting material. The purity of isolatedreticulocytes may be assessed using microscopy in that reticulocytes arecharacterized by a reticular (mesh-like) network of ribosomal RNA thatbecomes visible under a microscope with certain stains such as newmethylene blue or brilliant cresyl blue. Surface expression oftransferrin receptor (CD71) is also higher on reticulocytes anddecreases and they mature to erythrocytes, allowing for enrichment andanalysis of reticulocyte populations using anti-CD71 antibodies (See,e.g., Miltenyi CD71 microbeads product insert No. 130-046-201).Alternatively, analysis of creatine and hemoglobin A1C content andpyruvate kinase, aspartate aminotransferase, and porphobilinogendeaminase enzyme activity may be used to assess properties of theisolated reticulocytes relative to mature erythrocytes (See, e.g., Brunet al., Blood 76:2397-2403 (1990)). For example, the activity ofporphobilinogen deaminase is nearly 9 fold higher whereas the hemoglobinA1C content is nearly 10 fold less in reticulocytes relative to matureerythrocytes.

In some embodiments, cells suitable for generating syntheticmembrane-receiver complexes are differentiated ex vivo and/or in vivofrom one or more stem cells. In one embodiment, the one or more stemcells are one or more hematopoietic stem cells. Various assays may beperformed to confirm the ex vivo differentiation of culturedhematopoietic stem cells into reticulocytes and erythrocytes, including,for example, microscopy, hematology, flow cytometry, deformabilitymeasurements, enzyme activities, and hemoglobin analysis and functionalproperties (Giarratana et al., Nature Biotech. 23:69-74 (2005)). Thephenotype of cultured hematopoietic stem cells may be assessed usingmicroscopy of cells stained, for example, with Cresyl Brilliant blue.Reticulocytes, for example, exhibit a reticular network of ribosomal RNAunder these staining conditions whereas erythrocytes are devoid ofstaining. Enucleated cells may also be monitored for standardhematological variables including mean corpuscular volume (MCV;femtoliters (fL)), mean corpuscular hemoglobin concentration (MCHC; %)and mean corpuscular hemoglobin (MCH; pg/cell) using, for example, anXE2100 automat (Sysmex, Roche Diagnostics).

In some embodiments, the synthetic membrane-receiver complexes areassessed for their basic physical properties, e.g., size, mass, volume,diameter, buoyancy, density, and membrane properties, e.g., viscosity,deformability fluctuation, and fluidity.

In one embodiment, the diameter of the synthetic membrane-receivercomplexes is measured by microscopy or by automated instrumentation,e.g., a hematological analysis instrument. In one embodiment thediameter of the synthetic membrane-receiver complexes is between about1-20 microns. In one embodiment, the diameter of the syntheticmembrane-receiver complexes is at least in one dimension between about1-20 microns. In one embodiment, the diameter of the syntheticmembrane-receiver complexes is less than about 1 micron. In oneembodiment, the diameter of the complexes in one dimension is largerthan about 20 microns. In one embodiment, the diameter of the syntheticmembrane-receiver complexes is between about 1 micron and about 20microns, between about 2 microns and about 20 microns between about 3microns and about 20 microns between about 4 microns and about 20microns between about 5 microns and about 20 microns between about 6microns and about 20 microns, between about 5 microns and about 15microns or between about 10 microns and about 30 microns.

In one embodiment, the mean corpuscular volume of the syntheticmembrane-receiver complexes is measured using a hematological analysisinstrument. In one embodiment the volume of the mean corpuscular volumeof the complexes is greater than 10 fL, 20 fL, 30 fL, 40 fL, 50 fL, 60fL, 70 fL, 80 fL, 90 fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL,or greater than 150 fL. In one embodiment the mean corpuscular volume ofthe complexes is less than 30 fL, 40 fL, 50 fL, 60 fL, 70 fL, 80 fL, 90fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL, 160 fL, 170 fL, 180fL, 190 fL, 200 fL, or less than 200 fL. In one embodiment the meancorpuscular volume of the complexes is between 80-100 femtoliters (fL).

In one embodiment the average buoyant mass of the syntheticmembrane-receiver complexes (pg/cell) is measured using a suspendedmicrochannel resonatory or a double suspended microchannel resonatory(see e.g., Byun et al PNAS 2013 110(19):7580 and Bryan et al. Lab Chip2014 14(3):569).

In one embodiment the dry density of the synthetic membrane-receivercomplexes is measured by buoyant mass in an H2O-D2O exchange assay (seee.g., Feijo Delgado et al., PLOS One 2013 8(7):e67590).

In some embodiments, the synthetic membrane-receiver complexes have anaverage membrane deformability fluctuation of standard deviation greaterthan 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 mrad asmeasured by spatial light interference microscopy (SLIM) (see e.g.,Bhaduri et al., Sci Reports 2014, 4:6211).

In one embodiment, the average membrane viscosity of a population ofsynthetic membrane-receiver complexes is measured by detecting theaverage fluorescence upon incubation with viscosity-dependent quantumyield fluorophores (see e.g., Haidekker et al. Chem & Biol 20018(2):123).

In one embodiment, the membrane fluidity of the syntheticmembrane-receiver complexes is measured by fluorescence polarization,e.g., with BMG Labtech POLARstar Omega microplate reader.

For example, to measure deformability reticulocytes may be separatedfrom nucleated cells on day 15 of culture, for example, by passagethrough a deleukocyting filter (e.g., Leucolab LCG2, Macopharma) andsubsequently assayed using ektacytometry. The enucleated cells aresuspended in 4% polyvinylpyrrolidone solution and then exposed to anincreasing osmotic gradient from 60 to 450 mosM. Changes in the laserdiffraction pattern (deformability index) of the cells are recorded as afunction of osmolarity, to assess the dynamic deformability of the cellmembrane. The maximum deformability index achieved at a physiologicallyrelevant osmolarity is related to the mean surface area of erythrocytes.

In some embodiments, the synthetic membrane-receiver complexes areanalyzed for hemoglobin contents. Assays of hemoglobin may be used toassess the phenotype of differentiated cells (Giarratana et al., NatureBiotech. 23:69-74 (2005)). For example, high performance liquidchromatography (HPLC) using a Bio-Rad Variant II Hb analyzer (Bio-RadLaboratories) may be used to assess the percentage of various hemoglobinfractions. Oxygen equilibrium may be measured using a continuous methodwith a double-wavelength spectrophotometer (e.g., Hemox analyzer, TCS).The binding properties of hemoglobin may be assessed using flashphotolysis. In this method, the rebinding of CO to intracellularhemoglobin tetramers are analyzed at 436 nm after photolysis with a 10nanosecond pulse at 532 nm.

The synthetic membrane-receiver complexes described herein can bepurified following manufacture if desired. Many suitable methods ofpurification are known in the art. For example, the syntheticmembrane-receiver complexes are purified by centrifugation,magnetophoresis, irradiation, acoustophoresis, and chemical or physicalenucleation. In one embodiment synthetic membrane-receiver complexes arepurified by ex vivo maturation with, e.g., a stromal cell co-culture. Inone embodiment, synthetic membrane-receiver complexes are purified bychemical or enzymatic treatment of complexes, e.g by treatment with adeglycosylation enzyme.

In one embodiment the synthetic membrane-receiver polypeptide complexesare purified by disabling any residual replicative potential of themembrane-receiver polypeptide complexes. In one embodiment the syntheticmembrane-receiver polypeptide complexes are subjected to radiation,e.g., X rays, gamma rays, beta particles, alpha particles, neutrons,protons, elemental nuclei, UV rays in order to damage residualreplication-competent nucleic acids.

Ionizing radiation is energy transmitted via X rays, gamma rays, betaparticles (high-speed electrons), alpha particles (the nucleus of thehelium atom), neutrons, protons, and other heavy ions such as the nucleiof argon, nitrogen, carbon, and other elements. X rays and gamma raysare electromagnetic waves like light, but their energy is much higherthan that of light (their wavelengths are much shorter). Ultraviolet(UV) light is a radiation of intermediate energy that can damage cellsbut UV light differs from the forms of electromagnetic radiationmentioned above in that it does not cause ionization (loss of anelectron) in atoms or molecules, but rather excitation (change in energylevel of an electron). The other forms of radiation—particles—are eithernegatively charged (electrons), positively charged (protons, alpha rays,and other heavy ions), or electrically neutral (neutrons).

Radiation-induced ionizations may act directly on the cellular componentmolecules or indirectly on water molecules, causing water-derivedradicals. Radicals react with nearby molecules in a very short time,resulting in breakage of chemical bonds or oxidation (addition of oxygenatoms) of the affected molecules. The major effect in cells is DNAbreaks. Since DNA consists of a pair of complementary double strands,breaks of either a single strand or both strands can occur. However, thelatter is believed to be much more important biologically. Mostsingle-strand breaks can be repaired normally thanks to thedouble-stranded nature of the DNA molecule (the two strands complementeach other, so that an intact strand can serve as a template for repairof its damaged, opposite strand). In the case of double-strand breaks,however, repair is more difficult and erroneous rejoining of broken endsmay occur. These so-called misrepairs result in induction of mutations,chromosome aberrations, or cell death.

Deletion of DNA segments is the predominant form of radiation damage incells that survive irradiation. It may be caused by (1) misrepair of twoseparate double-strand breaks in a DNA molecule with joining of the twoouter ends and loss of the fragment between the breaks or (2) theprocess of cleaning (enzyme digestion of nucleotides—the componentmolecules of DNA) of the broken ends before rejoining to repair onedouble-strand break.

Radiations differ not only by their constituents (electrons, protons,neutrons, etc.) but also by their energy. Radiations that cause denseionization along their track (such as neutrons) are calledhigh-linear-energy-transfer (high-LET) radiation, a physical parameterto describe average energy released per unit length of the track. (Seethe accompanying figure.) Low-LET radiations produce ionizations onlysparsely along their track and, hence, almost homogeneously within acell. Radiation dose is the amount of energy per unit of biologicalmaterial (e.g., number of ionizations per cell). Thus, high-LETradiations are more destructive to biological material than low-LETradiations—such as X and gamma rays—because at the same dose, thelow-LET radiations induce the same number of radicals more sparselywithin a cell, whereas the high-LET radiations—such as neutrons andalpha particles—transfer most of their energy to a small region of thecell. The localized DNA damage caused by dense ionizations from high-LETradiations is more difficult to repair than the diffuse DNA damagecaused by the sparse ionizations from low-LET radiations.

In one embodiment, a population of synthetic membrane-receiverpolypeptide complexes are subjected to gamma irradiation using anirradiation dose of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, or more than 100 kGy.

In one embodiment, a population of synthetic membrane-receiverpolypeptide complexes are subjected to X-ray irradiation using anirradiation dose of more than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or greaterthan 10000 mSv.

The purity of a population of synthetic membrane-receiver complexes canbe assessed by measuring the homogeneity of the population. In oneembodiment, the average distribution width of the syntheticmembrane-receiver complexes is measured by a hematological analysisinstrument. In one embodiment, the population of syntheticmembrane-receiver complexes has a reticulocyte to non-reticulocyte ratiogreater than 10, 100, 1000, 10⁴, 10⁵, 10⁶, or greater than 10⁶. Thehomogeneity of the population of synthetic membrane-receiver complexesmay be assessed by measuring the stromal cell content of the population.In one embodiment, the population of synthetic membrane-receivercomplexes has less than 1 ppb of stromal cells. Alternatively or inaddition, the homogeneity of the population of syntheticmembrane-receiver complexes is assessed by measuring the viral titerand/or a bacterial colony forming potential of the population.

In one embodiment the homogeneity of a population of syntheticmembrane-receiver complexes is assessed by an in vitro assay. The invitro assays include those described herein for assessing the identityof the complexes, including but not limited to expansion rate, number,protein content or expression level, mRNA content or expression level,lipid content, partition of a substrate, catalytic activity, ormetabolic activity.

Mature erythrocytes for use in generating the syntheticmembrane-receiver complexes may be isolated using various methods suchas, for example, a cell washer, a continuous flow cell separator,density gradient separation, fluorescence-activated cell sorting (FACS),Miltenyi immunomagnetic depletion (MACS), or a combination of thesemethods (See, e.g., van der Berg et al., Clin. Chem. 33:1081-1082(1987); Bar-Zvi et al., J. Biol. Chem. 262:17719-17723 (1987); Goodmanet al., Exp. Biol. Med. 232:1470-1476 (2007)).

Erythrocytes may be isolated from whole blood by simple centrifugation(See, e.g., van der Berg et al., Clin. Chem. 33:1081-1082 (1987)). Forexample, EDTA-anticoagulated whole blood may be centrifuged at 800×g for10 min at 4° C. The platelet-rich plasma and buffy coat are removed andthe red blood cells are washed three times with isotonic saline solution(NaCl, 9 g/L).

Alternatively, erythrocytes may be isolated using density gradientcentrifugation with various separation mediums such as, for example,Ficoll, Hypaque, Histopaque, Percoll, Sigmacell, or combinationsthereof. For example, a volume of Histopaque-1077 is layered on top ofan equal volume of Histopaque-1119. EDTA-anticoagulated whole blooddiluted 1:1 in an equal volume of isotonic saline solution (NaCl, 9 g/L)is layered on top of the Histopaque and the sample is centrifuged at700×g for 30 min at room temperature. Under these conditions,granulocytes migrate to the 1077/1119 interface, lymphocytes, othermononuclear cells and platelets remain at the plasma/1077 interface, andthe red blood cells are pelleted. The red blood cells are washed twicewith isotonic saline solution.

Alternatively, erythrocytes may be isolated by centrifugation using aPercoll step gradient (See, e.g., Bar-Zvi et al., J. Biol. Chem.262:17719-17723 (1987)). For example, fresh blood is mixed with ananticoagulant solution containing 75 mM sodium citrate and 38 mM citricacid and the cells washed briefly in Hepes-buffered saline. Leukocytesand platelets are removed by adsorption with a mixture of α-celluloseand Sigmacell (1:1). The erythrocytes are further isolated fromreticulocytes and residual white blood cells by centrifugation through a45/75% Percoll step gradient for 10 min at 2500 rpm in a Sorvall SS34rotor. The erythrocytes are recovered in the pellet while reticulocytesband at the 45/75% interface and the remaining white blood cells band atthe 0/45% interface. The Percoll is removed from the erythrocytes byseveral washes in Hepes-buffered saline. Other materials that may beused to generate density gradients for isolation of erythrocytes includeOptiPrep™, a 60% solution of iodixanol in water (from Axis-Shield,Dundee, Scotland).

Erythrocytes may be separated from reticulocytes, for example, usingflow cytometry (See, e.g., Goodman el al., Exp. Biol. Med. 232:1470-1476(2007)). In this instance, whole blood is centrifuged (550×g, 20 min,25° C.) to separate cells from plasma. The cell pellet is resuspended inphosphate buffered saline solution and further fractionated onFicoll-Paque (1.077 density), for example, by centrifugation (400×g, 30min, 25° C.) to separate the erythrocytes from the white blood cells.The resulting cell pellet is resuspended in RPMI supplemented with 10%fetal bovine serum and sorted on a FACS instrument such as, for example,a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, N.J.,USA) based on size and granularity.

Erythrocytes may be isolated by immunomagnetic depletion (See, e.g.,Goodman, el al., (2007) Exp. Biol. Med. 232:1470-1476). In thisinstance, magnetic beads with cell-type specific antibodies are used toeliminate non-erythrocytes. For example, erythrocytes are isolated fromthe majority of other blood components using a density gradient asdescribed herein followed by immunomagnetic depletion of any residualreticulocytes. The cells are pre-treated with human antibody serum for20 min at 25° C. and then treated with antibodies against reticulocytespecific antigens such as, for example, CD71 and CD36. The antibodiesmay be directly attached to magnetic beads or conjugated to PE, forexample, to which magnetic beads with anti-PE antibody will react. Theantibody-magnetic bead complex is able to selectively extract residualreticulocytes, for example, from the erythrocyte population.

Erythrocytes may also be isolated using apheresis. The process ofapheresis involves removal of whole blood from a patient or donor,separation of blood components using centrifugation or cell sorting,withdrawal of one or more of the separated portions, and transfusion ofremaining components back into the patient or donor. A number ofinstruments are currently in use for this purpose such as for examplethe Amicus and Alyx instruments from Baxter (Deerfield, Ill., USA), theTrima Accel instrument from Gambro BCT (Lakewood, Colo., USA), and theMCS+9000 instrument from Haemonetics (Braintree, Mass., USA). Additionalpurification methods may be necessary to achieve the appropriate degreeof cell purity.

In some embodiments, the synthetic membrane-receiver complexes aredifferentiated ex vivo and/or in vivo from one or more reticulocytes.Reticulocytes may be used to generate synthetic membrane-receivercomplexes. Reticulocytes are immature red blood cells and composeapproximately 1% of the red blood cells in the human body. Reticulocytesdevelop and mature in the bone marrow. Once released into circulation,reticulocytes rapidly undergo terminal differentiation to matureerythrocytes. Like mature erythrocytes, reticulocytes do not have a cellnucleus. Unlike mature erythrocytes, reticulocytes maintain the abilityto perform protein synthesis. In some embodiments, exogenous nucleicacid (such as mRNA) encoding a receiver is introduced into reticulocytesto generate synthetic membrane-receiver complexes.

Reticulocytes of varying age may be isolated from peripheral blood basedon the differences in cell density as the reticulocytes mature.Reticulocytes may be isolated from peripheral blood using differentialcentrifugation through various density gradients. For example, Percollgradients may be used to isolate reticulocytes (See, e.g., Noble el al.,Blood 74:475-481 (1989)). Sterile isotonic Percoll solutions of density1.096 and 1.058 g/ml are made by diluting Percoll (Sigma-Aldrich, SaintLouis, Mo., USA) to a final concentration of 10 mM triethanolamine, 117mM NaCl, 5 mM glucose, and 1.5 mg/ml bovine serum albumin (BSA). Thesesolutions have an osmolarity between 295 and 310 mOsm. Five milliliters,for example, of the first Percoll solution (density 1.096) is added to asterile 15 ml conical centrifuge tube. Two milliliters, for example, ofthe second Percoll solution (density 1.058) is layered over the higherdensity first Percoll solution. Two to four milliliters of whole bloodare layered on top of the tube. The tube is centrifuged at 250×g for 30min in a refrigerated centrifuge with swing-out tube holders.Reticulocytes and some white cells migrate to the interface between thetwo Percoll layers. The cells at the interface are transferred to a newtube and washed twice with phosphate buffered saline (PBS) with 5 mMglucose, 0.03 mM sodium azide and 1 mg/ml BSA. Residual white bloodcells are removed by chromatography in PBS over a size exclusion column.

Alternatively, reticulocytes may be isolated by positive selection usingan immunomagnetic separation approach (See, e.g., Brun et al., Blood76:2397-2403 (1990)). This approach takes advantage of the large numberof transferrin receptors that are expressed on the surface ofreticulocytes relative to erythrocytes prior to maturation. Magneticbeads coated with an antibody to the transferrin receptor may be used toselectively isolate reticulocytes from a mixed blood cell population.Antibodies to the transferrin receptor of a variety of mammalianspecies, including human, are available from commercial sources (e.g.,Affinity BioReagents, Golden, Colo., USA; Sigma-Aldrich, Saint Louis,Mo., USA). The transferrin antibody may be directly linked to themagnetic beads. Alternatively, the transferrin antibody may beindirectly linked to the magnetic beads via a secondary antibody. Forexample, mouse monoclonal antibody 10D2 (Affinity BioReagents, Golden,Colo., USA) against human transferrin may be mixed with immunomagneticbeads coated with a sheep anti-mouse immunoglobulin G (Dynal/Invitrogen,Carlsbad, Calif., USA). The immunomagnetic beads are then incubated witha leukocyte-depleted red blood cell fraction. The beads and red bloodcells are incubated at 22° C. with gentle mixing for 60-90 min followedby isolation of the beads with attached reticulocytes using a magneticfield. The isolated reticulocytes may be removed from the magnetic beadsusing, for example, DETACHaBEAD® solution (from Invitrogen, Carlsbad,Calif., USA). Alternatively, reticulocytes may be isolated from in vitrogrowth and maturation of CD34+ hematopoietic stem cells using themethods described herein.

Terminally-differentiated, enucleated erythrocytes can be separated fromother cells based on their DNA content. In a non-limiting example, cellsare first labeled with a vital DNA dye, such as Hoechst 33342(Invitrogen Corp.). Hoechst 33342 is a cell-permeant nuclearcounterstain that emits blue fluorescence when bound to double-strandedDNA. Undifferentiated precursor cells, macrophages or other nucleatedcells in the culture are stained by Hoechst 33342, while enucleatederythrocytes are Hoechst-negative. The Hoechst-positive cells can beseparated from enucleated erythrocytes by using fluorescence activatedcell sorters or other cell sorting techniques. The Hoechst dye can beremoved from the isolated erythrocytes by dialysis or other suitablemethods.

A population of synthetic membrane-receiver complexes can be purified byreducing the nuclear material content of the population of complexes.For example, the enucleation rate of the population of complexes isincreased, and/or the number of enucleated synthetic membrane-receivercomplexes is increased or enriched.

Populations of synthetic membrane-receiver complexes can be incubatedwith a small molecule, e.g., an actin inhibitor, e.g., cytochalasin A,B, C, D, E, F, H, J, and then centrifuged to remove nuclear material.Alternatively or in addition, a population of syntheticmembrane-receiver complexes can be mechanically manipulated by passingthrough progressively smaller size-restrictive filters to remove nuclearmaterial. The population of synthetic membrane-receiver complexes mayalso be incubated on a fibronectin-coated plastic surface to increasethe removal of nuclear material. In one embodiment, the population ofsynthetic membrane-receiver complexes is incubated in co-culture withstromal cells, e.g., macrophages, to increase the removal of nuclearmaterial.

In some embodiments, the population of synthetic membrane-receivercomplexes is greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, orgreater than 99.9% enucleated.

In some embodiments, the synthetic membrane-receiver complexes are notco-cultured with support cells, e.g., with an adherent stromal layer. Insome embodiments, the population of synthetic membrane-receivercomplexes is generated by contacting erythroid cells with a receiver anddifferentiating the erythroid cells to obtain a population of enucleatedcells comprising the receiver. The population of syntheticmembrane-receiver complexes is obtained without an enrichment step, suchas gravitational separation, magnetic or fluorescent sorting,irradiation, poisoning of nucleated cells, and the like to select forenucleated cells.

In some embodiments, the population of synthetic membrane-receivercomplexes is comprised of greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%,99.9%, or greater than 99.9% of synthetic membrane-receiver complexesthat lack nuclear material as assessed by an assay to detect nuclearmaterial such as those described herein.

In some embodiments, the presence, biological activity and/or functionof a receiver, such as a receiver polypeptide exhibited by syntheticmembrane-receiver complexes is assessed. Many suitable assays areavailable and known in the art.

In one embodiment, the receiver is a polypeptide on the surface of thesynthetic membrane-receiver complex. The presence of the receiver can beassessed by assays including but not limited to flow cytometry, westernblotting, RT-PCR, Northern blotting, Coombs rosetting, massspectrometry. In one embodiment, the receiver is a polypeptide in theinterior of the synthetic membrane-receiver complex. The presence of thereceiver can be assessed by assays including but not limited to Westernblotting, RT-PCR, Norther blotting, PCR, Southern blotting, massspectrometry.

In one embodiment, the receiver is a nucleic acid on the surface of thesynthetic membrane-receiver complex. The presence of the receiver can beassessed by assays including but not limited to flow cytometry, flowcytometry with a homologous fluorescent probe, southern blotting,northern blotting, PCR. In one embodiment, the receiver is a nucleicacid in the interior of the synthetic membrane-receiver complex. Thepresence of the receiver can be assessed by assays including but notlimited to southern blotting, northern blotting, PCR.

In one embodiment, the receiver is a small molecule on the surface ofthe synthetic membrane-receiver complex. The presence of the receivercan be assessed by assays including but not limited to flow cytometry,mass spectrometry. In one embodiment, the receiver is a small moleculein the interior of the synthetic membrane-receiver complex. The presenceof the receiver can be assessed by assays including but not limited tomass spectrometry, fluorescence spectroscopy.

In one embodiment, the receiver is a lipid in the membrane of thesynthetic membrane-receiver complex. The presence of the receiver can beassessed by assays including but not limited to mass spectrometry, flowcytometry, membrane solubility, fluorescence polarization, spatial lightinterferences microscopy.

In one embodiment, the receiver is fluorescent or is fused to afluorescent molecule or is co-expressed from an exogenous nucleic acid(e.g., in a vector) with a fluorescent reporter protein like GFP. Thepresence of the receiver in or on the synthetic membrane-receivercomplex can be assessed by assays including but not limited to flowcytometry, fluorescence spectroscopy, absorbance spectroscopy.

In one embodiment, the receiver is a gaseous molecule. The presence ofthe receiver in or on the synthetic membrane-receiver complex can beassessed by assays including but not limited to chemiluminescenceassays, mass spectroscopy.

The presence of the receiver in or on the synthetic membrane-receivercomplex can be assessed by flow cytometry in a quantitative fashionusing calibration beads such as commercially available cytometrycalibration beads to quantify the number of receivers on an individualcomplex. Alternatively or in addition, the presence of the receiver inor on the synthetic membrane-receiver complex can be assessed by Westernblot in a quantitative fashion using a standard of known concentrationthat is detectable using the same detection reagents as the receiver,and in this way the number of receivers on an individual complex can bequantified.

In some embodiments, the presence of two or more different receivers canbe assessed by the same or different methods, either simultaneously, insequential fashion, or in parallel. For example, in one embodiment areceiver on the surface can be assessed by flow cytometry using anantibody specific to the receiver and a different receiver not on thesurface that is fluorescent can be assessed by fluorescent signal usinga different channel in flow cytometry. In a different example, areceiver on the surface can be assessed by flow cytometry and adifferent receiver not on the surface can be assessed by Western blot.

In a specific embodiment, the receiver is retained on the syntheticmembrane-receiver complex following terminal differentiation of the cellsource. For example, the membrane-receiver complex is generated from acultured erythroid cell and the expression or presence of the receiveris assessed following terminal differentiation of the cell by a suitablemethod, e.g., by flow cytometry, Western blot, immunoprecipitation,fluorescence spectroscopy, chemiluminescence, Southern blot, Northernblot, or absorbance spectroscopy.

In a specific embodiment, the receiver is retained on the syntheticmembrane-receiver complex following circulation in vivo afteradministration of the synthetic membrane-receiver complex to a subject.The synthetic membrane-receiver complex can be injected into alaboratory animal or animal model, such as a mouse intravenously, e.g.,via the tail vein, or is injected into a human intravenously. Then bloodis drawn and the presence of the receiver on the syntheticmembrane-receiver complex is assessed by suitable assay, e.g., by flowcytometry, Western blot, immunoprecipitation, fluorescence spectroscopy,chemiluminescence, Southern blot, Northern blot, or absorbancespectroscopy.

In some embodiments, the biological activity of the receiver in or onthe synthetic membrane-receiver complex, the overall biological activityof the complex, and the overall activity of a population of complexescan be assessed by in vitro assays.

In some embodiments, the activity of the synthetic membrane-receivercomplex is rapidly iterated using a model cell line. For example, alibrary of suitable receivers is expressed in a model cell line, e.g.,HEK293T or K562, and the activity is assessed via a suitable assay; thenthe best receiver candidate, e.g., the one that is expressed at thehighest level or one that demonstrates the highest activity in thesuitable assay, is expressed, e.g., in cultured erythroid cells togenerate synthetic membrane-receiver complexes.

In one embodiment, the activity of the synthetic membrane receivercomplex is rapidly iterated using a cultured mouse erythroid cell model.For example, a library of suitable receivers is expressed in culturedmouse erythroid cells; activity is assessed in a suitable mouse model ofdisease or a suitable mouse model system for assessing activity; thebest receiver candidate, e.g., the one that is expressed at the highestlevel or the one that demonstrates the highest activity in the suitableassay, is then expressed, e.g., in cultured erythroid cells to generatesynthetic membrane-receiver complex.

In some instances, the receiver is an enzyme and the activity of thereceiver can be assessed by an enzymatic assay in which thedisappearance of a specific substrate molecule is detected or theappearance of a specific product molecule is detected. Such assaysinclude but are not limited to, colorimetric assays, mass spectrometry,HPLC, fluorescent assays.

For example, a) the receiver is adenosine deaminase (ADA) and theenzymatic assay detects the conversion of adenosine to inosine; b) thereceiver is phenylalanine hydroxylase (PAH) and the assay detects theconversion of phenylalanine to tyrosine; c) the receiver isphenylalanine ammonia lyase (PAL) and the assay detects the conversionof phenylalanine to trans-cinnamic acid; d) the receiver is thymidinephosphorylase (TP) and the assay detects the conversion of thymidine tothymine and 2-deoxy-ribose; e) the receiver is Purine nucleosidephosphorylase (PNP) and the assay detects the conversion of inosine tohypoxanthine, adenosine into adenine, and guanosine into guanine; f) thereceiver is homogentisate 1,2-dioxygenase (HDG) and the assay detectsthe conversion of homogentisate to maleylacetoacetate; g) the receiveris cystathionine beta synthase and the assay detects the conversion ofserine and homocysteine to cystathionine; h) the receiver is oxalateoxidase and the assay detects the oxidation of oxalate.

In some embodiments, activity of the synthetic membrane-receiver complexis assessed in an animal model, for example a mouse model, andimmunodeficient mouse, or an NSG mouse, of a disease, for example ametabolic disease or an enzyme deficiency, or that can demonstrate theeffect of the synthetic membrane-receiver complex, for example a mouseinto which a substrate is injected and the product of thereceiver-mediated conversion measured.

In one embodiment, the receiver is complement receptor 1 (CR1)polypeptide, a derivative or functional fragment thereof. The activityof the CR1 receiver can be assessed in several ways including, forexample, the specific capture of immune complexes by the CR1 receiver,the efficient transfer of the immune complexes to macrophages, or the invivo clearance of immune complexes from a mouse.

Functionality of erythroid cells overexpressing CR1 receiver may beassessed by one or more processes: capture of immune complexes on theerythroid cell surface comprising CR1 receiver, release of the immunecomplexes to macrophages while sparing the erythroid cell comprising CR1receiver, and proper circulation of the erythroid cells comprising CR1receiver. These three parameters can be assayed in vitro Immune complexcapture assays are described in the art, e.g., Oudin et al., J Immunol2000 and Schifferli et al., J Immunol 1991. For example, labeled immunecomplexes are incubated with erythroid cells expressing native CR1 orCR1 receiver polypeptide or a fragment thereof and the number of immunecomplexes captured by the erythroid cells is assayed by flow cytometry.Macrophage transfer assays are described in the art, e.g., Kuhn et al.,J Immunol 1998. For example, labeled immune complexes loaded ontoerythrocytes expressing native CR1 or CR1 receiver polypeptide or afragment thereof are incubated with macrophages. The transfer of immunecomplex from erythrocyte surface to macrophage, and the consumption orsparing of erythrocytes by macrophages, can be measured by flowcytometry. Proper circulation can be predicted by analyzing erythroidcell morphology and deformability. Morphology of erythroid cellsexpressing native CR1 or CR1 receiver polypeptide or a fragment thereofcan be assessed by eye using standard microscopy techniques, asdescribed e.g., by Giarratana et al., Blood 2011 and Repik et al., ClinExp Immunol 2005. Deformability of erythroid cells expressing native CR1or CR1 receiver polypeptide or a fragment thereof can be assessed byektacytometry, also known as laser-assisted optical rotational cellanalysis (LORCA), as described e.g., Giarratana et al., Blood 2011.

For example, a synthetic membrane-CR1 receiver complex (the complexcomprises a CR1 polypeptide receiver) is incubated with immunecomplexes, such as in vivtro generated immune complexes orpatient-derived immune complexes. The capture of the immune complexes bythe CR1 receiver is assessed by, for example, flow cytometry using afluorescent marker in the immune complex or by flow cytometry using asecondary detection agent against an element of the immune complex.

In one embodiment, the synthetic membrane-CR1 receiver complex is firstincubated with immune complexes and then incubated with macrophages,such as primary macrophages, primary monocytes, cultured macrophages,cultured monocytpes, U937 cells, PMA-activated U937 cells, AA9 cells,RAW 264.7 cells, J774 Cells, THP1 cells, KG-1 cells, NR8383 cells,MV-4-11 cells, 3D4/31 cells, MD cells, Fcwf-4 cells, DH82 cells. Themacrophages are assayed by, for example, flow cytometry or radiography,for the presence of immune complexes transferred by the syntheticmembrane-CR1 receiver complex. The transfer of captured immune complexesfrom cultured erythroid cells to macrophages is a standard assay in theart, see for example: Repik et al. 2005 Clin Exp Immunol. 140:230; Li etal. 2010 Infection Immunity 78(7):3129.

In one embodiment, activity of the synthetic membrane-CR1 receivercomplex is assessed in an animal model. For example, a suitable mousemodel may be used, such as an immunodeficient mouse, or an NSG mouse.The mouse disease model can be for example an immune complex disease,such as lupus. Mouse models include NZBWF1/J, MRL/MpJ, MRL/MpJ-Fas(lpr),Smn.C3-Fasl/J, NZM2410/Aeg, 129S4-Cd48, Cg-Sle1, NZM-Sle1 Sle2Sle3/LmoJ, and BXSB.129P2. Alternatively or in addition, a diseasephenotype may be introduced into a mouse, e.g., by injection of immunecomplexes. The synthetic membrane-CR1 receiver complexes may be injectedinto any suitable mouse (or other animal model) to test one or morebiological effects of the complex, e.g., the clearance of the injectedimmune complexes by the synthetic membrane-CR1 receiver complex.

In some embodiments, the synthetic membrane-receiver complex comprisinga CR1 receiver is not generated in a mouse and/or are not generated frommouse erythroid cells. In some embodiments, the syntheticmembrane-receiver complex comprising a CR1 receiver is not generated ina laboratory animal and/or are not generated from an erythroid cellsderived from a laboratory animal.

In one embodiment, the receiver is a complement regulatory molecule orhas complement regulatory activity. This activity of the receiver can beassessed by both in vitro and in vivo assays. For instance, the activityof the receiver can be assessed by measuring the reduction in an invitro complement activation assay, e.g., CH50 assay that measurescomplement-mediated lysis of sensitized sheep erythroctyes, or AH50assay that measured alternate pathway complement-mediated lysis ofnon-sensitized rabbit erythrocytes. Alternatively, the activity of thereceiver can be assessed by detecting the cleavage or absence ofcleavage, which may or may not expose a neoepitope, of a recombinantcomplement component that has been incubated with the receiver,including but not limited to e.g., the cleavage of recombinant C2 intoC2a and C2b, the cleavage of factor B into factor Ba and factor Bb, thecleavage of factor C3b into iC3bH and iC3bL, the cleavage of C3bBb intoC3b and Bb, the cleavage of C4bBb into C4b and Bb, or the cleavage offactor C4b into iC4bH and iC4bL. The cleavage or absence of cleavage ofa suitable recombinant complement component can be assessed by proteinanalysis methods known in the art including, but not limited to, e.g.,chromatography, gel electrophoresis, ELISA, and western blotting.Suitable in vivo assays for receiver activity include injection of thesynthetic membrane-receiver complex into animal, for example a mouse,and examining the deposition of complement factors, for example membraneattack complex, by histological staining.

In one embodiment, the receiver is capable of binding or capturing atarget and the activity of the receiver can be assessed by detecting thecaptured target on the receiver in vitro or in vivo.

In one embodiment, the synthetic membrane-receiver complex is incubatedwith the target in vitro, and the capture of the target by the receiveris detectected using an in vitro assay including but not limited to, forexample, flow cytometry, immunohistochemistry, magnetic separation,radiography, colony-forming assays, microscopy.

In one embodiment, the synthetic membrane-receiver complex is incubatedwith the target in vitro, and the capture of the target by the receiveris detected using an in vitro co-culture assay including but not limitedto for example a macrophage consumption assay of opsonized receivercomplex, a T cell activation assay, a B cell stimulation assay, a mastcell degranulation assay, an infectious potential assay.

In an embodiment, the synthetic membrane-receiver complex is incubatedwith the target in vitro, and the release or off-rate of the capturedtarget is measured using an in vitro assay including but not limited to,for example, flow cytometry, immunohistochemistry, magnetic separation,radiography, colony-forming assays, microscopy.

The capture of the target by the synthetic membrane-receiver complex canbe assayed in an in vivo assay, for example in an animal, including amouse model of diseases in which the target is naturally present in themouse. Suitable diseases include bacterial infections, viral infections,fungal infections, immune complex diseases, self-antibody diseases,hyperlipidemia, hyperglycemia. In other mouse models, the target isadministered to the mouse externally, e.g., by injection or by feeding.In these assays, the capture of the target by the syntheticmembrane-receiver complex is assayed either by examining the animal, e.gthe plasma, the tissue, for reduction or retention of the target, or byisolating or collecting the receiver complex from the animal, e.g., fromthe blood, from the plasma, from a tissue, and assaying the presence ofthe target on the receiver using an in vitro assay including, but notlimited to, for example, flow cytometry, immunohistochemistry, magneticseparation, radiography, colony-forming assays, microscopy.

In some embodiments, the receiver is capable of binding or capturing atarget and substantially increasing the clearance of the target in vivo,or substantially reducing the concentration of the target incirculation. The activity of the receiver on the syntheticmembrane-receiver complex can be assessed by detecting the enhancedclearance of the target in vitro or in vivo.

In one embodiment, the synthetic membrane-receiver complex is incubatedwith the target in vitro, and the capture of the target by the receiveris detectected using an in vitro assay including but not limited to, forexample, flow cytometry, immunohistochemistry, magnetic separation,radiography, colony-forming assays, microscopy. Subsequently, thesynthetic membrane-receiver complex is incubated in a co-culture assaywith a cell known to promote clearance, for example a macrophage or amonocyte, and the clearance of the target and receiver complex isassessed by, for example, flow cytometry, immunohistochemistry, magneticseparation, radiography, colony-forming assays, microscopy.

In one embodiment, the synthetic membrane-receiver complex is incubatedwith the target in vitro, and the capture of the target by the receiveris detectected using an in vitro assay including but not limited to, forexample, flow cytometry, immunohistochemistry, magnetic separation,radiography, colony-forming assays, microscopy. Subsequently, thesynthetic membrane-receiver complex is incubated in a physical systemthat mimics the clearance mechanism of the complex in vivo, for examplean artificial spleen, a microchannel, a packed column, a resin, a tissueexplant, a centrifuge, and the clearance of the target and receivercomplex is assessed by, for example, flow cytometry,immunohistochemistry, magnetic separation, radiography, colony-formingassays, microscopy.

In one embodiment, the clearance of the target by the syntheticmembrane-receiver complex is assayed in an in vivo assay, for example inan animal, including, for example, a mouse model of diseases in whichthe target is naturally present in the mouse, for example bacterialinfection, viral infection, fungal infection, immune complex disease,self-antibody disease, hyperlipidemia, hyperglycemia, or for example, amouse model in which the target is administered to the mouse externally,e.g., by injection or by feeding. In these assays, the clearance of thetarget by the receiver complex is assayed either by examining theanimal, e.g the plasma, the tissue, for reduction of the target, or byisolating or collecting the synthetic membrane-receiver complex from theanimal, e.g., from the blood, from the plasma, from a tissue, andassaying the presence of the target on the receiver using an in vitroassay including, but not limited to, for example, flow cytometry,immunohistochemistry, magnetic separation, radiography, colony-formingassays, microscopy.

In some embodiments, the synthetic membrane-receiver complex is capableof delivering a suitable receiver to a specific subcellular compartment,for example a lysosome.

For example, a receiver may be delivered to the lysosomal compartment ofa target cell, e.g., a macrophage. The successful delivery of thereceiver to the lysosomal compartment of a target cell is assessed bymicroscopy and the detection of punctate spots corresponding to afluorescent receiver or fluorescent receiver detection agent.Alternatively or in addition, the successful delivery of the receiver tothe lysosomal compartment of a target cell is assessed by microscopy andthe colocalization of a fluorescent receiver detection agent with afluorescent detection agent for a known lysosomal marker, e.g.,lysotracker, LAMP-1.

In some embodiments, the receiver is an enzyme that can degrade toxiccomponents that have built up in the lysosome of a cell exhibiting thegenotype or phenotype of, or derived from a patient with, a lysosomalstorage disease. For example, the receiver is capable of degrading thetoxic material built up in the cell and rescue the cell phenotype, e.g.,preventing cell death.

The population of synthetic membrane-receiver complexes can be assessedfor the inability of the complexes to replicate, the ability of thecomplexes to circulate safely through the vasculature, and the lack ofimmunogenicity of the complexes.

In some embodiments, the safety of the population of syntheticmembrane-receiver complexes is assessed by measuring the replicationpotential of the population of complexes using a suitable in vitro or invivo assay. For example, tests for a substantial inability of thesynthetic membrane-receiver complexes to self-replicate include: a) asubstantial inability to form a tumor when injected into animmunocompromised mouse; b) a substantial inability to form a colonywhen cultured in soft agar; c) a substantial inability to incorporatethymidine in a thymidine incorporation assay; d) a substantial lack ofpositive signal upon transfection with DNA encoding a fluorescentreporter, e.g., less than 10%, 1%, 0.1%, 0.01%, 0.001%, 1 ppm, 100 ppb,10 ppb, 1 ppb, 100 ppt, 10 ppt, 1 ppt, or less than 1 ppt positivesignal; e) a substantial lack of positive signal upon staining with anuclear dye, e.g., less than 10%, 1%, 0.1%, 0.01%; and 0.001%, 1 ppm,100 ppb, 10 ppb, 1 ppb, 100 ppt, 10 ppt, 1 ppt, or less than 1 pptpositive signal; f) a substantial lack of positive signal upon stainingwith cell markers of hematological malignancy, e.g., CKIT, CD34, EpCam,e.g., less than 10%, 1%, 0.1%, 0.01%, 0.001%, 1 ppm, 100 ppb, 10 ppb, 1ppb, 100 ppt, 10 ppt, 1 ppt, or less than 1 ppt positive signal. Incertain embodiments, synthetic membrane-receiver complexes are providedthat do not contain a substantial amount of a replicating nucleic acid.

In some embodiments, the safety of the population of syntheticmembrane-receiver complexes is assessed by measuring the ability of anadministered complex to circulate in vivo (in the circulatory system ofa subject) without causing substantial vascular occlusion or inductionof the clotting cascade. Optionally, the circulation pharmacokinetics ofthe synthetic membrane-receiver complexes may be assessed.

In one embodiment, the circulation pharmacokinetics of the syntheticmembrane-receiver complexes is assessed by injecting the complex into ananimal intravenously, such as a mouse. The mouse can be an NSG(nod-SCID-gamma) immunodeficient mouse. The mouse is depleted ofmacrophages prior to injection with the complex, e.g., byintraperitoneal injection of human red blood cells, or by intravenousinjection with clodronate liposomes. The synthetic membrane-receivercomplexes can be labeled with a fluorescent dye, e.g., CFSE. Afterinjection of the complexes, blood is drawn and the number of syntheticmembrane-receiver complexes remaining is assessed by, e.g., flowcytometry, western blot, and the clearance rate of the syntheticmembrane-receiver complexes is deduced from these data. Human red bloodcells can be injected into the same animal model as the syntheticmembrane-receiver complexes and the clearance rates of the complexes andhuman red blood cells are compared.

In one embodiment, the risk of activation of the clotting cascade by thesynthetic membrane-receiver polypeptide complex is assessed with an invitro assay. In one embodiment, the synthetic membrane-receiverpolypeptide complex is incubated with human blood and clotting cascadeactivation is assessed by measuring the time required for coagulation inthe presence of kaolin, negatively-charged phospholipids, and calcium(activated partial thromboplastn time (aPTT) test), see e.g., Exner andRickard, Biomedicine 1977 27(2):62, or by measuring the time requiredfor coagulation in the presence of thromboplastin and calcium(prothrombin time (PT) test), see e.g., Jaques and Dunlop 1945, Am JPhysiol 145:67. The normal range for the aPTT test is approximately25-38 seconds. The normal range for the PT test is approximately 10-12seconds.

In one embodiment, any adverse events induced by the syntheticmembrane-receiver complexes are assessed by injecting the complex intoan animal intravenously and assessing the activation of the clottingcascade. The level of clotting cascade induction is assessed by drawingblood and assessing the levels of clotting cascade components in theplasma by, e.g., Western Blot or ELISA. The clotting cascade componentsare typically fibrinogen breakdown products, e.g., fibrinopeptide A andfibrinopeptide B. For example, the level of clotting cascade inductionis assessed by drawing blood and assessing the levels of clottingactivity in the plasma by platelet activation assay, e.g., incubatingthe plasma with platelets and assessing the activation of the plateletsby flow cytometry, e.g., by staining for markers of activation, e.g., bystaining for PAC-1, CD62p, or CD40L.

In one embodiment, any adverse events induced by the syntheticmembrane-receiver complexes are assessed by injecting the complex intoan animal intravenously and assessing the activation of inflammatorypathways. The level of inflammation can be assessed by drawing blood andassessing the levels of inflammatory cytokines in the plasma by, e.g.,Western Blot or ELISA. In one embodiment, the inflammatory citokines areinterferon gamma, tumor necrosis factor alpha, or IL-12 fragment p70.

In one embodiment, any adverse events induced by the syntheticmembrane-receiver complexes are assessed by injecting the complex intoan animal intravenously and assessing the status of tissues, e.g.,liver, spleen, heart, lungs, brain, skin, kidneys. The status of tissuecan be assessed by gross necropsy, dissection of the tissue,histological staining, and imaging by microscopy.

In one embodiment, the ability of the synthetic membrane-receivercomplex to circulate in vivo without causing substantial vascularocclusion or activation of the clotting cascade is assessed by measuringthe deformability of the complex. The deformability of the syntheticmembrane-receiver complex is assessed using an in vitro assay. Forexample, the assay is an osmotic fragility assay. Mechanical fragilityof the synthetic membrane-receiver complex can be assessed by measuringthe structural integrity in response to shear stress in a Couett-typeshearing system. In one embodiment, the deformability of the syntheticmembrane-receiver complex is assessed using an Ektacytometer. In oneembodiment, the deformability of the synthetic membrane-receiver complexis assessed by measuring the elongation index at a defined pressure bylaser diffraction using a laser-assisted optical rotational cellanalyzer (LORCA) instrument. In one embodiment, the deformability of thesynthetic membrane-receiver complex is assessed by measuring the transittime through a series of micron-scale constrictions of defineddimensions at a fixed pressure in a microfluidic device. In oneembodiment, the deformability of the synthetic membrane-receiver complexis assessed by measuring the survival rate through a series ofmicron-scale constrictions of defined dimensions at a fixed pressure ina microfluidic device. The microfluidic device can be selected from oneof the following, including but not limited to, a poly dimethyl siloxane(PDMS) chip with micron-scale constrictions (e.g., Hoelzle et al. J VisExp 2014 91:e51474); a chip with funnel-shaped constrictions (e.g., Guoet al. Lab Chip 2012 12:1143); a PDMS chip with pillars (e.g., Zhang etal. PNAS 2012 109(46):18707); or an in vitro artificial spleen microbeadpacked column (Guillaume DePlaine et al., Blood 2011, 117(8)).

In one embodiment, the ability of the synthetic membrane-receivercomplex to circulate in vivo without causing substantial vascularocclusion or activation of the clotting cascade is assessed by measuringthe vascular occlusion of the complex. Vascular occlusion of thesynthetic membrane-receiver complex can be assessed using an in vitroassay. For example, the vascular occlusion of the syntheticmembrane-receiver complex is assessed using an ex vivo assay. Thesynthetic membrane-receiver complex is incubated at a 1:1 ratio withreference human red blood cells and induction of multi-cell rosettes areassessed by light microscopy in comparison to a reference assay withRh-mismatched blood. The vascular occlusion of the syntheticmembrane-receiver complex is assessed by measuring the adhesion of thecomplex to human vascular endothelial cells under flow conditions, seee.g., Kaul D K, Finnegan E, and Barabino G A (2009) Microcirculation16(1):97-111. Alternatively or in addition, vascular occlusion isassessed by measuring the peripheral resistance unit (PRU) increase inan ex vivo perfusion assay of rat vascular endothelium, see e.g., Kaul,Fabry and Nagel, PNAS 1989, 86:3356. Further, vascular occlusion isassessed by intravital microscopy, see e.g., Zennadi et al. 2007 Blood110(7):2708. Vascular occlusion may also be assessed by measuring flowrates and adhesion of the complex in vitro graduated height flowchambers, see e.g., Zennadi et al 2004, Blood 104(12):3774.

In some embodiments, the safety of the population of syntheticmembrane-receiver complexes is assessed by measuring the immunogenicityof the population of complexes using a suitable in vitro or in vivoassay.

For example, the population of synthetic membrane-receiver complexes a)does not induce agglutination in a Coombs test using serum from theintended recipient subject or b) does not induce agglutination in aCoombs test using pooled human serum.

In one embodiment, the population of synthetic membrane-receivercomplexes is derived from a progenitor cell that has been genotyped forthe predominant blood group antigens and matched to the blood groupantigen genotype of the recipient.

In one embodiment, the population of synthetic membrane-receivercomplexes comprises a receiver or other exogenous protein that has lessthan 10%, 1%, 0.1%, 0.01%, 0.001%, or less than 0.001% predicted T cellreactivity by an in silico T cell epitope prediction algorithm.

In one embodiment, the population of synthetic membrane-receivercomplexes comprises a receiver or other exogenous protein that has lessthan 10%, 1%, 0.1%, 0.01%, 0.001%, or less than 0.001% reactivity in anin vitro T cell activation assay, e.g., Antitope Inc. EpiScreen assay.

For example, synthetic membrane-receiver complexes derived fromerythrocytes can be centrifuged and resuspended in appropriate solution(e.g., standard AS-3 solution) for infusion into subjects in needthereof. In some embodiments, the synthetic membrane-receiver complexesto be infused have the same ABO type as that of the recipient tominimize the risk of infusion-associated immune reactions. The syntheticmembrane-receiver complexes can also be pretreated to remove bloodtype-specific antigens or otherwise reduce antigenicities. Methodssuitable for this purpose include, but are not limited to, thosedescribed in U.S. Patent Application Publication Nos. 20010006772 and20030207247.

Methods of Treatment and Prevention

Provided herein are methods of modulating the circulatory concentrationof a target to treat or prevent a disease, disorder or conditionassociated with the presence, absence, elevated or depressedconcentration of the target in the circulatory system of a subject. Thesubject may suffer from the disease, disorder or condition or may be atrisk of developing the disease, disorder or condition. The methodsprovided herein include the administration of a suitable syntheticmembrane-receiver polypeptide complex described herein in an amounteffective to substantially modulate the circulatory concentration of thetarget, thereby preventing or treating the disease, disorder orcondition. In some embodiments, the synthetic membrane-receiverpolypeptide complex is formulated as a pharmaceutical composition. Insome embodiments, the pharmaceutical composition is formulated forintravenous injection to the subject. The compositions may beadministered once to the subject. Alternatively, multipleadministrations may be performed over a period of time. For example,two, three, four, five, or more administrations may be given to thesubject. In some embodiments, administrations may be given as needed,e.g., for as long as symptoms associated with the disease, disorder orcondition persist. In some embodiments, repeated administrations may beindicated for the remainder of the subject's life. Treatment periods mayvary and could be, e.g., no longer than a year, six months, threemonths, two months, one month, two weeks, one week, three days, twodays, or no longer than one day.

In some embodiments, the compositions are administered at least twiceover a treatment period such that the disease, disorder or condition istreated, or a symptom thereof is decreased. In some embodiments, thecompositions are administered at least twice over a treatment periodsuch that the disease, disorder or condition is treated, or a symptomthereof is prevented. In some embodiments, the pharmaceuticalcomposition is administered a sufficient number of times over atreatment period such that the circulatory concentration of the targetis substantially decreased during the treatment period. In someembodiments, the pharmaceutical composition is administered a sufficientnumber of times over a treatment period such that the circulatoryconcentration of the target self-antibody is substantially decreasedduring the treatment period such that one or more symptoms of theself-antibody mediated disease, disorder or condition is prevented,decreased or delayed. In some embodiments, decreasing the circulatoryconcentration of the target includes decreasing the peak concentration,while in others it includes decreasing the average concentration. Insome embodiments, a substantial decrease during the treatment period canbe determined by comparing a pretreatment or post-treatment period inthe human subject, or by comparing measurements made in a populationundergoing treatment with a matched, untreated control population. Insome embodiments, the circulatory concentration of the target isdecreased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, 99.99%, or greater than 99.99% during part or theentirety of the treatment period. In some embodiments, the circulatoryconcentration of the target is decreased by at least about 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or greater than99.99% within about 1, 5, 10, 15, 20, 30, 40, or 50 minutes, or about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, or 23 hours, or 1, 2, 3, 4, 5, or 6 days or about 1, 2, 3, 4, 5, or6 weeks of the administration.

In some embodiments, the pharmaceutical composition is administered asufficient number of times over a treatment period such that thecirculatory concentration of the target is decreased at a rate greaterthan i) the endogenous clearance rate of the target\by the humansubject, or ii) the endogenous production rate of the target by thehuman subject, or iii) both i) and ii). In some embodiments, thepharmaceutical composition is administered a sufficient number of timesa treatment period such that the circulatory concentration of the targetis substantially decreased for at least about one week, two weeks, threeweeks, four weeks, one month, two months, three months, four months,five months, six months, or greater than six months. In someembodiments, the pharmaceutical composition is administered a sufficientnumber of times a treatment period such that the circulatoryconcentration of the target is substantially decreased for a period oftime at least as long as the treatment period.

In some embodiments, the pharmaceutical composition is administered at afrequency sufficient to effectively reduce the circulatory concentrationof the target below a level that is associated with a symptom of thedisease, disorder or condition.

In some embodiments, the time interval between administrations within atreatment period is no longer than the period in which the number ofsynthetic membrane-receiver complexes in circulation is reduced to lessthan about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of syntheticmembrane-receiver complexes present in the administered pharmaceuticalcomposition.

Diseases, disorders and conditions associated with targets in thecirculatory system that may be treated or prevented by administeringsynthetic membrane-receiver polypeptide complexes are described herein.

Diseases, disorders and conditions associated with targets in thecirculatory system that may be treated or prevented by administeringsynthetic membrane-receiver polypeptide complexes include, but are notlimited to: self-antibody-mediated diseases, complementdysregulation-associated diseases, immune complex associated diseases,amyloidoses, diseases associated with infectious agents or pathogens(e.g., bacterial, fungal, viral, parasitic infections), diseaseassociated with toxic proteins, diseases associated with theaccumulation of lipids, diseases associated with apoptotic, necrotic,aberrant or oncogenic mammalian cells, and metabolic diseases.

Provided herein, in some embodiments, are methods for the treatment orprevention of diseases or conditions that are associated with targets(e.g., molecules or entities) that reside, at least in part, in thecirculatory system. The methods comprise, in certain embodiments,administering to a subject in need thereof functional erythroid cellscomprising a receiver, populations of functional erythroid cellscomprising a receiver, or compositions, preferably pharmaceuticalcompositions comprising functional erythroid cells comprising areceiver, in an amount effective to treat or prevent the disease orcondition that is associated with molecules or entities that reside, atleast in part, in the circulatory system.

Methods are provided for the treatment or prevention of inflammation anddiseases associated with inflammation, including sepsis, autoimmunedisease, cancer, and microbial infections, the methods comprising,administering to a subject in need thereof an erythrocyte comprising animmune-modulatory receiver in an amount effective to treat or preventthe inflammation or an associated disease. In some embodiments, anerythrocyte comprises an immunomodulatory receiver that comprises achemokine or cytokine receptor. In a particular embodiment, thechemokine receptor is DARC.

Methods are provided for the modulation of chemokine homeostasis atsites of inflammation, the methods comprising, administering to asubject in need thereof an erythrocyte comprising a chemokine-modulatoryreceiver in an amount effective to modulate chemokine homeostasis atsites of inflammation. In some embodiments, the erythrocyte comprising achemokine-modulatory receiver comprises a receiver that comprises achemokine receptor. In a particular embodiment, the chemokine receptoris DARC. In some embodiments, the site of inflammation is vascular.(Darbonne, J Clinical Invet, 1991).

Further provided are methods of inducing toxin clearance. The methodsinclude administering to a subject in need thereof a population offunctional erythroid cells comprising a receiver that is capable ofinteracting with a toxin, such as e.g., an antibody, scFv or nanobodyreceiver, in an amount effective to clear toxins from circulation. Suchmethods may be employed to sequester the toxin and reduce the amount oftissue damage that would otherwise occur within the vasculature anddissipating its pathogenic effects in a less acute manner.

In some embodiments, provided are methods of treating diseases,including, but not limited to, metabolic diseases, cancers, clotting andanti-clotting diseases. The methods include administering to a subjectin need thereof a pharmaceutical composition of functional erythroidcells comprising a receiver provided herein in an amount sufficient totreat the metabolic disease, the cancer, the clotting disease oranti-clotting disease of the subject.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes exhibit one or more receiver polypeptides on their surface,exposed to the environment the circulatory system of the subject.

In other embodiments, the synthetic membrane-receiver polypeptidecomplexes comprise one or more receiver polypeptides facing theunexposed side of the synthetic membrane-receiver polypeptide complex.

The receiver polypeptide may interact with targets that are present inthe circulatory system. The interaction of the receiver with the targetmay include, but is not limited to: i) binding of the receiver to thetarget; ii) degrading the target, iii) cleaving the target; iv)sequestering the target, and/or v) catalytically converting the target.

For example, the receiver polypeptide may be an antibody, a single-chainvariable fragment, a nanobody, a diabody, a darpin, a lyase, ahydrolase, a protease, or a nuclease.

In some embodiments, the synthetic membrane-receiver complex comprises areceiver that is not a polypeptide. In some embodiments, the receivercomprises a carbohydrate (e.g., a GAG), a lipid, DNA, a RNA, a peptidenucleic acid (PNA), or a non-protein ligand, drug or substrate that iscapable of interacting with the target.

In some embodiments, the interaction between the receiver and the targetleads to a direct or indirect reduction of the concentration of thetarget in the circulatory system. For example, a receiver may directlyconvert a target into a different product. The receiver may have acatalytic activity toward the target, such as cleaving, degrading, etc.The conversion may be from a target to a degradation product, from atoxic or harmful target to a non-toxic product, etc. The catalyticactivity of the receiver may also involve addition of one or morechemical groups to the target. Modification of the target by thereceiver may cause, directly or indirectly, e.g., de/phosphorylation,de/ubiquitination, de/methylation, glycosylation, etc. on the target.The receiver may indirectly cause a second modification on the target ifthe receiver put a first modification that leads to a secondmodification, e.g., by a different enzyme. Any such modifications maythen lead, e.g., to the degradation of the target or to a conversion ofthe target to a non-harmful or less harmful product. In someembodiments, a decrease in target concentration may be directly orindirectly associated with an increase in a non-target compound. Forexample, a toxic metabolite may be converted into a non-toxic or lesstoxic metabolite. In another example, a target may be converted into aproduct that is lacking or is present in an depressed amount. In thiscase, a disease, disorder or condition may be associated with the lackof or depressed amount of the non-target compound and conversion of thetarget, e.g., by a catalytic action of the synthetic membrane-receiverpolypeptide complex is capable of increasing the amount of thenon-target compound effective to treat the disease, disorder orcondition.

In other examples, the receiver may bind to the target and keep itsequestered, e.g., being associated with the synthetic membrane-receiverpolypeptide complex. The sequestration may inhibit an activity harboredby the target, e.g., a harmful activity, such as that exerted by aself-antibody which may be causative of an autoimmune disease, or abacterial toxin that may cause sepsis, etc. Alternatively or inaddition, upon sequestration or binding of the target the target isredistributed in the circulatory system of the subject according to thedistribution of the synthetic membrane-receiver polypeptide complex.This may significantly limit the volume of distribution of the target,and thus potentially its harmful or adverse impact. The target may bedegraded or accumulated at a specific site or organ in the body of thesubject directed by the turnover or half-life and distribution of thesynthetic membrane-receiver polypeptide complex.

The administration of the pharmaceutical composition may be sufficientto substantially decrease the concentration or amount of the targetmolecule in circulation during the treatment period, wherein thesubstantial decrease can be determined in comparison to a pre-treatmentor post-treatment period in the human subject, or via comparison ofmeasurements made in a population undergoing treatment as compared to amatched untreated control population. The substantial decrease of thetarget molecule can include a substantial decrease of the peakconcentration or amount of the target molecule present in a humanpatient or a substantial decrease in the average concentration or amountof the target molecule present in a human patient.

In some embodiments, provided are methods for treating diseases that aremarked by periodic flares, wherein a flare is defined as a recurrence ofsymptoms or an onset of more severe symptoms. Diseases marked byperiodic flares include self-antibody mediated diseases, immune complexassociated diseases, autoimmune diseases, including for example lupus,rheumatoid arthritis, and goodpasture syndrome.

In some embodiments, provided are methods comprising administering thepharmaceutical composition to a patient a sufficient number of timesover a treatment period such that the time between flares is reducedcompared to an individual who does not receive the pharmaceuticalcomposition.

In some embodiments, provided are methods comprising administering thepharmaceutical composition to a patient a sufficient number of timesover a treatment period such that the severity of the flares is reducedcompared to an individual who does not receive the pharmaceuticalcomposition.

In some embodiments, methods of treatment and prevention using syntheticmembrane-receiver complexes generated from erythroid cells describedherein do not comprise the step of detecting the erythroid cell in vivo,e.g., through a detection agent that is associated with the erythroidcell.

In some embodiments, the synthetic membrane-receiver complex is notgenerated from a human donor pluripotent hematopoietic stem cell. Insome embodiments, a population of synthetic membrane-receiver complexesis not expanded in a bioreactor. In some embodiments, the population ofsynthetic membrane-receiver complexes after expansion and/ordifferentiation does not comprise a single species of differentiatedhuman blood cells. In some embodiments, the synthetic membrane-receivercomplex is not a differentiated, mature human blood cell. In someembodiments, the synthetic membrane-receiver complex is not generatedfrom a blood cell derived from a universal donor, e.g. blood type O, Rhfactor negative.

In some embodiments, a synthetic membrane-ADA polypeptide receivercomplex is not used to treat severe combined immune deficiency(ADA-SCID).

In some embodiments, the methods of treatments described herein do notcomprise administering a synthetic membrane-receiver complex generatedfrom an erythroid cell that is contacted with a polypeptide receiver inan amount effective to induce immune tolerance to the polypeptidereceiver in a subject.

Suitable targets include biological compounds, inorganic or organiccompounds. Suitable targets may range in size from less than 100 Da,less than 250 Da, less than 500 Da, less than 1000 Da to targets of morethan 1 kDa. Targets can be, e.g., polypeptides, lipids, carbohydrates,nucleic acids, small molecules, metabolites and elements. In someembodiments, the target is an antibody, a complement factor, an immunecomplex, a serum amyloid protein, a bacterial pathogen, a fungalpathogen, a viral pathogen, or an infected, pathogenic, apoptotic,necrotic, aberrant or oncogenic mammalian cell.

Diseases, disorders and conditions associated with targets in thecirculatory system that may be treated or prevented by administeringsynthetic membrane-receiver polypeptide complexes include, but are notlimited to: self-antibody-mediated diseases, complementdysregulation-associated diseases, immune complex associated diseases,amyloidoses, diseases associated with infectious agents or pathogens(e.g., bacterial, fungal, viral, parasitic infections), diseaseassociated with toxic proteins, diseases associated with theaccumulation of lipids, diseases associated with apoptotic, necrotic,aberrant or oncogenic mammalian cells, and metabolic diseases.

In some embodiments, provided are methods of treating diseases,including, but not limited to, metabolic diseases, cancers, clotting andanti-clotting diseases. The methods include administering to a subjectin need thereof a pharmaceutical composition of erythrocyte cellscomprising a receiver provided herein in an amount sufficient to treatthe metabolic disease, the cancer, the clotting disease or anti-clottingdisease of the subject.

Self-Antibody Mediated Diseases

In some embodiment, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases,disorders or conditions that are associated with self-antibodies.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target self-antibody in a subject (e.g.,a human) suffering from or at risk of developing a self-antibodymediated disease, disorder or condition. The methods includeadministering a pharmaceutical composition comprising a syntheticmembrane-receiver polypeptide complex described herein. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the targetself-antibody. In certain embodiments, the administration is carried outintravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds and sequesters a target self-antibody that is present in thecirculatory system of the subject.

In certain embodiments, the pharmaceutical composition will reduce thetarget self-antibody load in the circulatory system, thereby reducingthe burden of the disease, disorder or condition associated with thepresence or elevated concentration of the target self-antibody. Diseasesassociated with target self-antibodies include, but are not limited to,Goodpasture syndrome, membranous glomerulonephropathy, antiphospholipidsyndrome (APS), catastrophic antiphospholipid syndrome (CAPS), and thoselisted in table 6 and table 8.

Self-antibody mediated diseases arise from an abnormal immune responseof the body against substances and tissues normally present in the body.This may be restricted to certain organs (e.g., in autoimmunethyroiditis) or involve a particular tissue in different places, e.g.,Goodpasture syndrome, which may affect the basement membrane in both thelung and the kidney. The treatment of self-antibody mediated diseasestypically includes immunosuppressive medications that decrease theimmune response, such as cyclophosphamide and rituximab. In certainembodiments, treatment with the pharmaceutical compositions describedherein is combined with one or more immunosuppressive medications, andeffective agents may be, e.g., co-administered or co-formulated.

In healthy subjects, the immune system is able to recognize and ignorethe body's own healthy proteins, cells, and tissues, and does notoverreact to non-threatening substances in the environment, such asfoods. If the immune system ceases to recognize one or more of thebody's normal constituents as “self” it may produce pathologicalself-antibodies, i.e. antibodies that recognize “self” antigens. Theseself-antibodies are directed against the body's own healthy cells,tissues, and/or organs, and may cause inflammation and tissue damage.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are provided that comprise receivers comprising epitopescapable of being recognized by target self-antibodies. For example, thetarget self-antibody may specifically recognizes glycoprotein (GP Ib-IX,IIb-IIIa, IV, or Ia-IIa), the NC1 domain of collagen α3 (IV), B2glycoprotein-1, or phospholipase A2 receptor, and the receiverpolypeptide may comprise an antigenic polypeptide selected from thegroup.

Target self-antibodies sequestered by the synthetic membrane-receiverpolypeptide complexes may be cleared from circulation, e.g., through thereticulo-endothelial system. Sequestration and/or degradation of thetarget self-antibody may reduce the degree of inflammation that isnormally caused when the self-antibody interacts with “self” tissues.

In one embodiment the disease or condition is antiphospholipid syndrome,the receiver is beta2-glycoprotein-1 or fragment thereof, and the targetis pathogenic self-antibody against beta2-glycoprotein-1.

In one embodiment the disease or condition is catastrophicantiphospholipid syndrome, the receiver is beta2-glycoprotein-1 orfragment thereof, and the target is pathogenic self-antibody againstbeta2-glycoprotein-1.

In one embodiment the disease or condition is cold agglutinin disease,the receiver is I/i antigen or fragment thereof, and the target ispathogenic self-antibody against I/i antigen.

In one embodiment the disease or condition is Goodpasture syndrome, thereceiver is a3 NC1 domain of collagen (IV) or fragment thereof, and thetarget is pathogenic self-antibody against a3 NC1 domain of collagen(IV).

In one embodiment the disease or condition is immune thrombocytopeniapurpura, the receiver is platelet glycoproteins (Ib-IX, IV, Ia-IIa) orfragment thereof, and the target is pathogenic self-antibody againstplatelet glycoprotein.

In one embodiment the disease or condition is membranous nephropathy,the receiver is phospholipase A2 receptor or fragment thereof, and thetarget is pathogenic self-antibody against phospholipase A2 receptor.

In one embodiment the disease or condition is warm antibody hemolyticanemia, the receiver is glycophorin A, glycophorin B, and/or glycophorinC, Rh antigen or fragment thereof, and the target is pathogenicself-antibody against glycophorins and/or Rh antigen.

Exemplary self-antibody diseases are Goodpasture syndrome, catastrophicantiphospholipid syndrome, and membranous glomerulopathy.

1. Goodpasture Syndrome

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for Goodpasture Syndrome.Subjects suffering from or at risk of developing Goodpasture Syndromemay be administered a pharmaceutical composition comprising thesynthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising α3IV collagen (COL4A3), or a derivativeor functional fragment thereof. A suitable receiver may be exhibited onthe surface of the synthetic membrane-receiver polypeptide complex.COL4A3 is normally found on kidney cells and presents a target to whichself-antibodies associated with Goodpasture syndrome have been shown tobind.

COL4A3 is found in air sacs in the lungs and glomeruli of the kidneys.Self-antibodies associated with Goodpasture syndrome are directedagainst the glomerular basement membrane and can cause kidney damage.Where the disorder is triggered by a viral respiratory infection or byintake of hydrocarbon solvents the resulting immune response can causebleeding in the air sacs of the lungs and inflammation in the kidney'sglomeruli.

2. Catastrophic Antiphospholipid Syndrome (CAPS)

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for antiphospholipid syndrome(APS). Subjects suffering from or at risk of developing APS may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising β2-glycoprotein 1 (b2GPI), or aderivative or functional fragment thereof. A suitable receiver may beexhibited on the surface of the synthetic membrane-receiver polypeptidecomplex. b2GPI is normally found on endothelial cells and presents atarget to which self-antibodies associated with APS have been shown tobind.

Antiphospholipid syndrome (APS) is a multisystem self-antibody mediatedcondition characterized by vascular thrombosis and/or pregnancy lossassociated with persistently positive antiphospholipid antibodies (aPL).Catastrophic APS (CAPS) is the most severe form of APS with multipleorgan involvement developing over a short period of time, usuallyassociated with microthrombosis. ‘Definite’ and ‘probable’ CAPS havebeen defined based on the preliminary classification criteria; however,aPL-positive patients with multiple organ thromboses and/or thromboticmicroangiopathies are encountered who do not fulfill these criteria.Previous APS diagnosis and/or persistent clinically significant aPLpositivity is of great importance for the CAPS diagnosis; however,almost half of the patients who develop CAPS do not have a history ofaPL positivity.

3. Membranous Glomerulopathy (MGN)

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for membranous glomerulopathy(MGN), also called membranous glomerulonephritis membranous nephritis(MN). Subjects suffering from or at risk of developing MGN may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising phospholipase A2 receptor, or aderivative or functional fragment thereof. A suitable receiver may beexhibited on the surface of the synthetic membrane-receiver polypeptidecomplex. Phospholipase A2 receptor is normally found on podocytes andpresents a target to which self-antibodies associated with MGN have beenshown to bind.

The term membranous nephritis, or membranous glomerulonephritis, is usedto describe a chronic glomerular disease that on light,immunofluorescence, and electron microscopy study of renal tissue showsa set of distinct morphologic features in glomeruli, including thickenedglomerular basement membrane (GBM) and GBM spikes, granular staining forIgG and complement along the periphery of glomerular all capillaryloops, and electron-dense subepithelial deposits corresponding to thegranular IgG staining.

Clinically, most patients present with nephrotic syndrome or haveproteinuria detected on a routine urinalysis. Idiopathic MN occurs inall age groups and races and both sexes all over the world and is aleading cause of nephrotic syndrome among Caucasian adults. Spontaneousremission of the disease is common in children but also occurs inadults. Although several immunosuppressive drugs often are used to treatindividual patients, with or without treatment, nearly a third ofpatients progress to end-stage renal disease.

Complement Dysregulation-Associated Diseases

In some embodiment, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases,disorders or conditions that are associated with complementdysregulation.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target complement protein in a subject(e.g., a human) suffering from or at risk of developing a disease,disorder or condition associated with complement dysregulation. Themethods include administering a pharmaceutical composition comprising asynthetic membrane-receiver polypeptide complex described herein. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the targetcomplement protein. In certain embodiments, the administration iscarried out intravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds and sequesters a target complement protein that is present in thecirculatory system of the subject.

In certain embodiments, the therapeutic compositions of the inventionprovide functional erythroid cells comprising receivers in compositionsthat are useful to treat, prevent, or reduce the severity of a disease,disorder or condition associated with complement pathophysiology orimproper immune complex clearance.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target complement protein in a subject(e.g., a human) suffering from or at risk of developing a disease,disorder or condition associated with complement dysregulation. Themethods include administering a pharmaceutical composition comprising asynthetic membrane-receiver polypeptide complex described herein. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the targetcomplement protein. In certain embodiments, the administration iscarried out intravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds and sequesters a target complement protein that is present in thecirculatory system of the subject.

Provided are therapeutic compositions present in an amount effect totreat a disease or condition associated with complement over-activationsuch as systemic lupus erythematosus, ischemia reperfusion injury, organtransplantation, myocardial infarction, rheumatoid arthritis,scleroderma, polyarteritis nodosa, serum sickness, arthus reaction,farmer's lung, Henoch-Schonlein purpura, bacterial endocarditis,vasculitis, and other Type III Hypersensitivity conditions. Furtherprovided are therapeutic compositions present in an amount able to treatan infectious disease in which opsonized pathogen is present in theblood, such as carbapenem-resistant enterobacteriaceae, drug resistantNeisseria gonorrhoeae, fully resistant streptococcus pneumonia, drugresistant tuberculosis, generalized bacterial sepsis, humanimmunodeficiency virus infection, hepatitis B virus infection, ormalaria. In a further embodiment, provided are therapeutic compositionspresent in an amount effect to treat a complement factordeficiency-associated disease such as cofactor H deficiency, paroxysmalnocturnal hemoglobinuria, factor B deficiency, factor D deficiency, C1qdeficiency, C1r deficiency, C4 deficiency, C2 deficiency, C3 deficiency,C5 deficiency, C6 deficiency, C7 deficiency, factor I deficiency, factorD deficiency, MBL deficiency, MASP2 deficiency, CD55 deficiency, CD59deficiency, and other deficiencies in genes associate with complementactivity including but not limited to those listed in table 6 and table8.

In certain embodiments, the pharmaceutical composition will reduce thetarget complement protein load in the circulatory system, therebyreducing the burden of the disease, disorder or condition associatedwith the presence or elevated concentration of the target complementprotein. Diseases associated with complement dysregulation include, butare not limited to, atypical hemolytic uremic syndrome (aHUS),paroxysmal nocturnal hemoglobinuria (PNH), age-related maculardegeneration (AMD), complement factor I (CFI) deficiency and thoselisted in table 6 and table 8.

In certain embodiments, the receiver polypeptide may specificallyinteract with a complement protein selected from the group consistingof: C1q, C1r, C1s, C2, C3, C3a, C3b, C4, C5, C5a, C5b, C6, C7, C8, andC9, Factor B, Factor D, Properdin, iC3b, C3c, C3dg, C3dk, C3e, Bb, C4a,C4b, and in table 5 and table 10.

In certain embodiments, the receiver polypeptide may comprise CD46,CD55, CD59, factor H, CR1, factor I, CR1, CR2, CR3, CR4, C3aR, C3eR,Decay-accelerating factor (DAF), Membrane cofactor protein (MCP), C3Beta chain Receptor, C1 inhibitor, C4 binding protein, and those listedin table 10.

Homologous restriction factormicrobial protein NalP, microbial proteinSpeB, microbial protein EspP, a derivative or a functional fragmentthereof. Alternatively or in addition, the receiver polypeptide maycomprise one or more complement control protein (CCP) modules and/orshort consensus repeats (SCR) of different origin.

The complement system is composed of more than 32 proteins including 7serum and 9 membrane regulatory proteins, 1 serosal regulatory protein,and 8 cell membrane receptors that bind complement fragments. Activationof complement occurs with the initiation of an inflammatory reaction,most of which occurs in the intravascular space. The soluble componentsof complement are present in the circulation and also in body fluids andtissues. In addition to the specific activation induced byantigen-antibody complexes, complement is activated through the patternrecognition receptors, which have the ability to discriminate betweenself and non-self antigens based on repeating patterns of molecularstructure (pathogen-associated molecular patterns) present on thesurface of pathogens. Complement-activating pattern recognitionreceptors include mannose-binding lectin (MBL), ficolins, C-reactiveprotein, C1q, and natural IgM (IgM).

Excessive, deregulated, or chronic inflammation can initiate orcontribute to several pathologies. For example, the activation ofcomplement during an inflammatory reaction contributes toinflammation-driven tissue injury, which occurs in theischemia/reperfusion (I/R) setting, vasculitides of various etiologies,nephritis, and arthritis. A deficiency in complement components may alsoresult in tissue injury, as observed in autoimmune reactions. Further,alterations in the expression of complement regulatory proteins may leadto excessive complement activation and can also contribute to tissueinjury.

In one embodiment the disease or condition is age-related maculardegeneration, the receiver is a suitable complement regulatory proteinor fragment thereof, and the target is active complement.

In one embodiment the disease or condition is atypical hemolytic uremicsyndrome, the receiver is complement factor H, or a suitable complementregulatory protein or fragment thereof, and the target is activecomplement.

In one embodiment the disease or condition is Complement Factor Ideficiency, the receiver is Complement Factor I, a suitable complementregulatory protein or fragment thereof, and the target is activecomplement.

In one embodiment the disease or condition is paroxysmal nocturnalhemoglobinuria, the receiver is a suitable complement regulatory proteinor fragment thereof, and the target is active complement.

In one embodiment the disease or condition is autoimmune hemolyticanemia, the receiver is a suitable complement regulatory molecule orfragment thereof, and the target is active complement.

in one embodiment the disease or condition is non-alcoholicsteatohepatitis, the receiver is a suitable complement regulatorymolecule or fragment thereof, and the target is active complement.

1. Paroxysmal Nocturnal Hemoglobinuria (PNH)

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for paroxysmal nocturnalhemoglobinuria (PNH). Subjects suffering from or at risk of developingPNH may be administered a pharmaceutical composition comprising thesynthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising a complement regulatory protein, such ascofactor H, or a derivative or functional fragment thereof. A suitablereceiver may be exhibited on the surface of the syntheticmembrane-receiver polypeptide complex and may be administered to reduceinflammation.

Paroxysmal nocturnal hemoglobinuria is an acquired disorder that leadsto the premature death and impaired production of blood cells. Thedisorder affects erythrocytes, leukocytes), and platelets(thrombocytes). PNH affects both sexes equally and can occur at any age,although it is most often diagnosed in young adulthood.

People with paroxysmal nocturnal hemoglobinuria have sudden, recurringepisodes of symptoms (paroxysmal symptoms), which may be triggered bystresses on the body, such as infections or physical exertion. Duringthese episodes, red blood cells are prematurely destroyed (hemolysis).Affected individuals may pass dark-colored urine due to the presence ofhemoglobin (hemoglobinuria). In many, but not all cases, hemoglobinuriais most noticeable in the morning, upon passing urine that hasaccumulated in the bladder during the night (nocturnal).

The premature destruction of red blood cells results in a deficiency ofthese cells in the blood (hemolytic anemia), which can cause signs andsymptoms such as fatigue, weakness, abnormally pale skin (pallor),shortness of breath, and an increased heart rate. People with PNH mayalso be prone to infections due to a deficiency of white blood cells.

Abnormal platelets associated with PNH can cause problems in the bloodclotting process. As a result, people with this disorder may experienceabnormal blood clotting (thrombosis), especially in large abdominalveins; or, less often, episodes of severe bleeding (hemorrhage).

2. Atypical Hemolytic Uremic Syndrome (aHUS)

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for atypical hemolytic uremicsyndrome (aHUS). Subjects suffering from or at risk of developing aHUSmay be administered a pharmaceutical composition comprising thesynthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising a complement regulatory protein, such ascofactor I, or a derivative or functional fragment thereof. A suitablereceiver may be exhibited on the surface of the syntheticmembrane-receiver polypeptide complex and may be administered to reduceinflammation.

Atypical hemolytic uremic syndrome (aHUS) is a rare syndrome ofhemolysis, thrombocytopenia, and renal insufficiency. Genetic mutationsin the alternate pathway of complement is the cause in more than 60% ofpatients affected by this thrombotic microangiopathy. aHUS may betreated using plasma therapy, complement blockade, and/or livertransplantation. Because aHUS shares many of the presentingcharacteristics of the other thrombotic microangiopathies, andconfirmatory genetic results are not available at the time ofpresentation, the diagnosis relies heavily on the recognition of aclinical syndrome consistent with the diagnosis in the absence of signsof an alternate cause of thrombotic microangiopathy.

3. Age-Related Macular Degeneration (AMD)

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for age related maculardegeneration (AMD). Subjects suffering from or at risk of developing AMDmay be administered a pharmaceutical composition comprising thesynthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising a complement regulatory protein, such asCD55 and CD59, or a derivative or functional fragment thereof. Asuitable receiver may be exhibited on the surface of the syntheticmembrane-receiver polypeptide complex and may be administered to reduceinflammation.

Age related macular degeneration (AMD) is a common form of blindness inthe western world and genetic variations of several complement genes,including the complement regulator Factor H, the central complementcomponent C3, Factor B, C2, and also Factor I confer a risk for thedisease. However deletion of a chromosomal segment in the Factor H genecluster on human chromosome 1, which results in the deficiency of theterminal pathway regulator CFHR1, and of the putative complementregulator CFHR3 has a protective effect for development of AMD. TheFactor H gene encodes two proteins Factor H and FHL1 which are derivedfrom alternatively processed transcripts. In particular a sequencevariation at position 402 of both Factor H and FHL1 is associated with arisk for AMD. A tyrosine residue at position 402 represents theprotective and a histidine residue the risk variant. AMD is considered achronic inflammatory disease, which can be caused by defective andinappropriate regulation of the continuously activated alternativecomplement pathway. This activation generates complement effectorproducts and inflammatory mediators that stimulate further inflammatoryreactions. Defective regulation can lead to formation of immunedeposits, drusen and ultimately translate into damage of retinal pigmentepithelial cells, rupture of the interface between these epithelialcells and the Bruch's membrane and vision loss.

Immune Complex-Associated Diseases

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases,disorders or conditions that are associated with immune complexes orimproper immune complex clearance.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target immune complex in a subject (e.g.,a human) suffering from or at risk of developing a disease, disorder orcondition associated with immune complexes. The methods includeadministering a pharmaceutical composition comprising a syntheticmembrane-complement receptor 1 (CR1) receiver complex. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the target immunecomplex. In certain embodiments, the administration is carried outintravenously.

In certain embodiments, synthetic membrane-complement receptor 1 (CR1)receiver complexes are administered that specifically bind and sequestera target immune complex that is present in the circulatory system of thesubject.

In some embodiments, functional erythroid cells comprising a receiverthat comprises complement receptor 1 (CR1) may be administered to asubject exhibiting immune complexes in circulation. For example, apopulation of functional erythroid cells comprising a receiver thatcomprises complement receptor 1 (CR1) can bind C3b within an immunecomplex and removal and clearance from circulation can occur through theliver.

Compositions comprising erythrocyte-bound CR1 receiver, such as aplurality of functional erythroid cells comprising elevated levels ofCR1, is preferably administered to a subject having been diagnosed withor being suspected of having a disease state that has resulted from anoverabundance of immune complex formation or that has caused a reductionor depletion in the native CR1 level, such as an immunecomplex-associated disorder or disease.

In certain embodiments, the pharmaceutical composition will reduce thetarget immune complex load in the circulatory system, and/or prevent thedeposition of immune complexes in sensitive soft tissue, therebyreducing the burden of the disease, disorder or condition associatedwith the presence or elevated concentration of the target immunecomplex. Diseases associated with complement dysregulation include, butare not limited to, systemic lupus erythematosus (SLE), lupus nephritis,IgA nephropathy, Dense Deposit Disease, lupus nephritis, Goodpasture'ssyndrome, membranoproliferative glomerulonephritis, immune complexvasculitis, cold agglutinin disease, polymyositis, acute pulmonaryhemorrhage, membranous glomerulonephritis, membranousglomerulonephritis, rapidly-progressive glomerulonephritis,post-streptococcal glomerulonephritis, post-staphylococcalglomerulonephritis, Pauci-immune glomerulonephritis, bloodhyperviscosity syndrome, and cutaneous leukocytoclastic angiitis andthose listed in table 6 and table 8.

In certain embodiments, the target immune complex comprises i) IgM orIgG, and ii) C3b and/or C4b.

In certain embodiments, the CR1 receiver comprises one or morecomplement control protein (CCP) modules, short consensus repeats (SCR)and/or long homologous repeats (LHRs). In some embodiments, the CR1receiver comprises a functional fragment of the full-length CR1polypeptide.

Type III, or immune-complex, reactions are characterized by tissuedamage caused by the activation of complement in response toantigen-antibody (immune) complexes (IgG and IgM) that are deposited intissues. Once the antigen-antibody complexes form, they are deposited invarious tissues of the body, especially the blood vessels, kidneys,lungs, skin, and joints. Deposition of the immune complexes causes aninflammatory response, which leads to the release of tissue-damagingenzymes and interleukin-1, which induces fever. Immune complexesunderlie many autoimmune diseases, such as systemic lupus erythematosus(an inflammatory disorder of connective tissue), most types ofglomerulonephritis (inflammation of the capillaries of the kidney), andrheumatoid arthritis.

Type III hypersensitivity reactions can be provoked by inhalation ofantigens into the lungs. A number of conditions are attributed to thistype of antigen exposure, including farmer's lung, caused by fungalspores from moldy hay; pigeon fancier's lung, resulting from proteinsfrom powdery pigeon dung; and humidifier fever, caused by normallyharmless protozoans that can grow in air-conditioning units and becomedispersed in fine droplets in climate-controlled offices. In each case,the person will be sensitized to the antigen with IgG antibodies to theagent circulating in the blood. Inhalation of the antigen will stimulatethe reaction and cause chest tightness, fever, and malaise, symptomsthat usually pass in a day or two but recur when the individual isre-exposed to the antigen. Permanent damage is rare unless individualsare exposed repeatedly. Some occupational diseases of workers who handlecotton, sugarcane, or coffee waste in warm countries have a similarcause, with the sensitizing antigen usually coming from fungi that growon the waste rather than the waste itself.

In one embodiment the disease or condition is IgA nephropathy, thereceiver is Complement receptor 1 or fragment thereof, and the target isImmune complexes.

In one embodiment the disease or condition is lupus nephritis, thereceiver is Complement receptor 1 or fragment thereof, and the target isimmune complex.

In one embodiment the disease or condition is systemic lupuserythematosus, the receiver is Complement receptor 1 or fragmentthereof, and the target is immune complex.

1. Systemic Lupus Erythematosus (SLE)

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for systemic lupus erythematosus(SLE). Subjects suffering from or at risk of developing SLE may beadministered a pharmaceutical composition comprising the syntheticmembrane-CR1 receiver complex to treat or prevent disease.

In certain embodiments, the CR1 receiver interacts with the target C3b,a constituent of a circulating immune complex. In some embodiments, theimmune complex once bound to the synthetic membrane-CR1 receiver complexis degraded through the reticulo-endothelial system.

Systemic lupus erythematosus (SLE) is a chronic inflammatory diseasethat has protean manifestations and follows a relapsing and remittingcourse. More than 90% of cases of SLE occur in women, frequentlystarting at childbearing age. SLE is a chronic autoimmune disease thatcan affect almost any organ system; thus, its presentation and courseare highly variable, ranging from indolent to fulminant. Inchildhood-onset SLE, there are several clinical symptoms more commonlyfound than in adults, including malar rash, ulcers/mucocutaneousinvolvement, renal involvement, proteinuria, urinary cellular casts,seizures, thrombocytopenia, hemolytic anemia, fever, andlymphadenopathy. In adults, Raynaud pleuritis and sicca are twice ascommon as in children and adolescents. A presentation of a triad offever, joint pain, and rash in a woman of childbearing age should promptinvestigation into the diagnosis of SLE.

SLE is an autoimmune disorder characterized by multisystem inflammationwith the generation of self-antibodies. Self-antibodies may be presentfor many years before the onset of the first symptoms of SLE. Further, Tcells from patients with lupus show defects in both signaling andeffector function (e.g., decreased secretion of interleukin (IL)-2).T-cell abnormalities offer targets for therapy, e.g., belimumab, whichtargets the B-lymphocyte stimulator (BLys) signaling pathway.

Many clinical manifestations of SLE are mediated by circulating immunecomplexes that form with antigens in various tissues or the directeffects of antibodies to cell surface components Immune complexes formin the microvasculature, leading to complement activation andinflammation. Moreover, antibody-antigen complexes deposit on thebasement membranes of skin and kidneys. In active SLE, this process hasbeen confirmed by demonstration of complexes of nuclear antigens such asDNA, immunoglobulins, and complement proteins at these sites.Self-antibodies (e.g., lupus anticoagulant (LA), and anti-ribosomal Pantibodies) can be used as biomarkers to determine futureneuropsychiatric events in SLE.

Other indications include the presence of serum antinuclear antibodies(ANAs) which are found in nearly all individuals with active SLE.Antibodies to native double-stranded DNA (dsDNA) are relatively specificfor the diagnosis of SLE. Cytotoxic T cells and suppressor T cells(which would normally down-regulate immune responses) are decreased. Thegeneration of polyclonal T-cell cytolytic activity is impaired. Helper(CD4⁺) T cells are increased. A lack of immune tolerance is observed inanimal lupus models.

2. IgA Nephropathy

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for IgA nephropathy. Subjectssuffering from or at risk of developing IgA nephropathy may beadministered a pharmaceutical composition comprising the syntheticmembrane-CR1 receiver complex to treat or prevent disease.

In certain embodiments, the CR1 receiver interacts with the target C3b,a constituent of a circulating IgA immune complex. In some embodiments,the immune complex once bound to the synthetic membrane-CR1 receivercomplex is degraded through the reticulo-endothelial system.

IgA nephropathy also known as Berger's disease is a kidney diseaseassociated with the accumulation of IgA-immune complexes. The presenceof the immune complexes triggers a local inflammation that reduces thekidneys' ability to filter waste, excess water and electrolytes from theblood. Kidney damage may be indicated by blood and protein in urine,high blood pressure and swollen feet.

IgA nephropathy usually progresses slowly over many years. Some subjectspresent blood in their urine without developing problems, someeventually achieve complete remission, and others develop end-stagekidney failure.

IgA nephropathy is the most common glomerulonephritis worldwide.Clinically, it is characterized by hematuria and proteinuria; about20-30% of the IgAN patients develop progressive renal failure within10-20 years from the onset of disease. Histologically, the glomerularmesangium contains deposits of IgA1, the C3 component of complement, andless frequently, IgG and/or IgM. Circulating immune complexes (CICs)composed of IgA1, C3, and IgG are involved in the pathogenesis of thedisease.

Serum IgA1 from IgAN patients may exhibit alterations in the glycan sidechains. Human IgA1 contains N- and O-linked glycans. IgA1 from IgANpatients display altered glycan moieties, usually with a reduced contentof galactose (Gal). The Gal-deficient IgA1 may be present in CICs withIgG. IgA1 from sera of IgAN patients exhibit increased binding tolectins specific for a terminal GalNAc, such as Helix aspersa (HAA) orHelix pomatia (HPO).

Amyloidoses

In some embodiment, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent amyloidosis.In some embodiments, membrane-receiver complexes are used that do notcontain a receiver polypeptide. The receiver can for example be aglycosaminoglycans (GAG).

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target serum amyloid protein in a subject(e.g., a human) suffering from or at risk of developing amyloid plaques.The methods include administering a pharmaceutical compositioncomprising a synthetic membrane-receiver complex described herein. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the target serumamyloid protein. In certain embodiments, the administration is carriedout intravenously.

In certain embodiments, synthetic membrane-receiver complexes areadministered that comprise a receiver that specifically binds,sequesters and/or degrades a target serum amyloid protein or targetamyloid plaque that is present in the circulatory system of the subject.

In certain embodiments, the pharmaceutical composition will reduce thetarget serum amyloid protein or amyloid plaque load in the circulatorysystem, e.g., preventing their deposition in soft tissue, therebyreducing the burden of the amyloidosis. Amyloidoses include, but are notlimited to, AA amyloidosis, light chain (AL) amyloidosis, beta-2microglobulin amyloidosis and those listed in table 6 and table 8.

In certain embodiments, the receiver polypeptide may specificallyinteract with a target serum amyloid protein selected from the groupconsisting of: amyloid P protein, amyloid A protein, light chain,misfolded transthyretin, and fibrinogen alpha chain.

Amyloidosis is a rare disease associated with amyloid plaques build up.Amyloidosis can affect different organs such as, e.g., the heart,kidneys, liver, spleen, nervous system and digestive tract. Severeamyloidosis can lead to life-threatening organ failure.

Acquired systemic amyloidosis is thought to be the cause of death inabout 1 in 1,000 persons in Western countries and is most common in theelderly. Systemic AL amyloidosis is the most common and serious type,accounting for over 60% of cases. Dialysis-related β₂-microglobulinamyloidosis affects about 1 million patients worldwide. Seniletransthyretin (ATTR) amyloidosis, which predominantly involves theheart, occurs in about one quarter of persons older than 80 years.

In one embodiment the disease or condition is AA amyloidosis, thereceiver is an an antibody-like binder to serum amyloid A protein orserum amyloid P component or fragment thereof, and the target is serumamyloid A protein and amyloid placques.

In one embodiment the disease or condition is beta2 microglobulinamyloidosis, the receiver is an an antibody-like binder to beta-2microglobulin or serum amyloid P component or fragment thereof, and thetarget is beta-2 microglobulin or amyloid placques.

In one embodiment the disease or condition is light chain amyloidosis,the receiver is an an antibody-like binder to light chain, serum amyloidP component or fragment thereof, and the target is antibody light chainor amyloid placques.

1. AA Amyloidosis

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for AA amyloidosis. Subjectssuffering from or at risk of developing AA amyloidosis may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complexes described herein to treat orprevent disease.

AA amyloidosis is a complication of chronic infections and inflammatorydiseases or any condition that leads to long-term overproduction of theacute phase reactant SAA. The amyloid fibrils are composed of anN-terminal cleavage fragment of SAA (the AA protein). AA amyloidosisoccurs in 1% to 5% of patients with rheumatoid arthritis, juvenileidiopathic arthritis and Crohn's disease. Tuberculosis and leprosy arealso important causes of AA amyloidosis in some parts of the world. Mostpatients present with proteinuria, and liver and gastrointestinalinvolvement may occur with time.

2. AL Amyloidosis

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for AL amyloidosis. Subjectssuffering from or at risk of developing AL amyloidosis may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complexes described herein to treat orprevent disease.

Systemic AL occurs in about 2% of people with monoclonal B-celldyscrasias. AL fibrils are derived from monoclonal immunoglobulin lightchains, affecting usually the kidneys, heart, liver and peripheralnerves.

3. β2-Microglobulin Amyloidosis

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for β2-Microglobulin amyloidosis.Subjects suffering from or at risk of developing β2-Microglobulinamyloidosis may be administered a pharmaceutical composition comprisingthe synthetic membrane-receiver polypeptide complexes described hereinto treat or prevent disease.

β₂-Microglobulin amyloid deposition occurs in patients withdialysis-dependent chronic renal failure, mainly affecting articular andperiarticular structures. It typically causes arthralgia of theshoulders, knees, wrists and small joints of the hand; joint swellingand carpal tunnel syndrome. The amyloid fibril precursor protein isβ₂-microglobulin, which is the invariant chain of the majorhistocompatibility complex (MHC) class I molecule and is expressed byall nucleated cells. Since it is normally filtered freely at theglomerulus, reabsorbed and catabolized by proximal tubular cells,decreasing renal function causes a proportionate rise in itsconcentration. Disease-related amyloidosis (DRA) is present in 20% to30% of patients within 3 years of starting dialysis for end-stage renalfailure.

In some embodiments, membrane-receiver complexes that do not contain areceiver polypeptide are used for treatment of an amyloidosis and or forthe reduction of a serum amyloid protein or amyloid plaque. In oneembodiment, the synthetic membrane-receiver complex comprises a receivercomprising a glycosaminoglycans (GAG), or a derivative or functionalfragment thereof. A suitable receiver may be exhibited on the surface ofthe synthetic membrane-receiver complex and may be administered to binda circulating amyloidogenic precursors. In certain embodiments, amyloiddeposits are prevented from forming.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising a serum amyloid P-component (SAP), or aderivative or functional fragment thereof. A suitable receiver may beexhibited on the surface of the synthetic membrane-receiver polypeptidecomplex and may be administered to prevent amyloids from aggregating.Serum amyloid P-component (protein SAP) has been described to bind invitro to isolated amyloid fibrils of both primary and secondary types.

Infectious Agent-Mediated Diseases and Conditions

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases,disorders or conditions that are associated with infectious agents.

In some embodiments, functional erythroid cells comprising a receiverspecific for circulating pathogens are administered to a subject in needthereof in an amount effective to treat an infectious disease in whichopsonized pathogen is present in the blood, such as carbapenem-resistantenterobacteriaceae, drug resistant Neisseria gonorrhoeae, fullyresistant Streptococcus pneumoniae, drug resistant tuberculosis,generalized bacterial sepsis, human immunodeficiency virus infection,hepatitis B virus infection, or malaria. In some embodiments, functionalerythroid cells comprise a receiver specific for circulating pathogensthat include, but are not limited to, the targets in table 5.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target infectious agent in a subject(e.g., a human) suffering from or at risk of developing an infectiousdisease. The methods include administering a pharmaceutical compositioncomprising a synthetic membrane-receiver polypeptide complex describedherein. The pharmaceutical composition is administered in an amounteffective to substantially reduce the circulatory concentration of thetarget infectious agent. In certain embodiments, the administration iscarried out intravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds, sequesters, and/or degrades an infectious agent, such as abacterium, a virus, a fungus, or a parasite that is present in thecirculatory system of the subject.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered to reduce the plasma titer of the infectiousagent, e.g., virus titer.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered to reduce the ability of the infectious agentto access enough host cells per unit of time. A decrease in the rate ofinfection of host cells may correlate with an increasing inability ofthe infectious agent to perpetuate the infection or perpetuate thedeleterious effect to the subject host. The infection may be suppressedand/or contained.

In certain embodiments, the pharmaceutical composition will reduce thetarget infectious agent load in the circulatory system, slowing orstopping the infection and aiding the immune system in its defense,thereby reducing the burden of the infectious disease. Infectiousdiseases include, but are not limited to, Hepatitis A, Hepatitis B,Hepatitis C, HIV, Ebola, C. difficile, C. botulinum, Anthrax, E. coli,mycobacterium tuberculosis, Candida, malaria and those listed in table 6and table 8.

In one embodiment the disease or condition is Anthrax (B. anthracis)infection, the receiver is an antibody-like binder to B. anthracissurface protein or fragment thereof, and the target is B. anthracis.

In one embodiment the disease or condition is C. botulinum infection,the receiver is an antibody-like binder to C. botulinum surface proteinor fragment thereof, and the target is C. botulinum.

In one embodiment the disease or condition is C. difficile infection,the receiver is an antibody-like binder to C. difficile surface proteinor fragment thereof, and the target is C. difficile.

In one embodiment the disease or condition is Candida infection, thereceiver is an antibody-like binder to candida surface protein orfragment thereof, and the target is candida.

In one embodiment the disease or condition is E. coli infection, thereceiver is an antibody-like binder to E. coli surface protein orfragment thereof, and the target is E. coli.

In one embodiment the disease or condition is Ebola infection, thereceiver is an antibody-like binder to Ebola surface protein or fragmentthereof, and the target is Ebola.

In one embodiment the disease or condition is Hepatitis B (HBV)infection, the receiver is an antibody-like binder to HBV surfaceprotein or fragment thereof, and the target is HBV.

In one embodiment the disease or condition is Hepatitis C (HCV)infection, the receiver is an antibody-like binder to HCV surfaceprotein or fragment thereof, and the target is HCV.

In one embodiment the disease or condition is Human immunodeficiencyvirus (HIV) infection, the receiver is an antibody-like binder to HIVenvelope proteins or CD4 or CCR5 or or fragment thereof, and the targetis HIV.

In one embodiment the disease or condition is M. tuberculosis infection,the receiver is an antibody-like binder to M. tuberculosis surfaceprotein or fragment thereof, and the target is M. tuberculosis.

In one embodiment the disease or condition is malaria (P. falciparum)infection, the receiver is an antibody-like binder to P. falciparumsurface protein or fragment thereof, and the target is P. falciparum.

1. Bacterial Infections

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for a bacterial infection.Subjects suffering from or at risk of developing a bacterial infectionmay be administered a pharmaceutical composition comprising thesynthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In some embodiments, the target is a bacterium. In certain embodiments,the target comprises a bacterial antigen. In some embodiments, thebacterial antigen comprises a cell surface antigen, a secreted toxin, ora secreted bacterial antigen.

Bacteremia is the presence of bacteria in the blood. Gram-negativebacteremia secondary to infection usually originates in thegenitourinary system or GI tract, or the skin in patients with decubitusulcers. Chronically ill and immunocompromised patients have an increasedrisk of gram-negative bacteremia. They may also develop bacteremia withgram-positive cocci, anaerobes, and fungi. Staphylococcal bacteremia iscommon in injection drug users. Bacteroides bacteremia may develop inpatients with infections of the abdomen and the pelvis, particularly thefemale genital tract. The bacteria most likely to cause bacteremiainclude members of the Staphylococcus, Streptococcus, Pseudomonas,Haemophilus, and Esherichia coli (E. coli) genera.

Bacterial infectious diseases that can be treated by the pharmaceuticalcompositions comprising a synthetic membrane-receiver polypeptidecomplex described herein include, but are not limited to, Mycobacteria,Rickettsia, Mycoplasma, Neisseria meningitides, Neisseria gonorrheoeae,Legionella, Vibrio cholerae, Streptococci, Staphylococcus aureus,Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynobacteriadiphtheriae, Clostridium spp., enterotoxigenic Eschericia coli, andBacillus anthracis. Other pathogens for which bacteremia has beenreported include: Rickettsia, Bartonella henselae, Bartonella quintana,Coxiella burnetii, chlamydia, Mycobacterium leprae, Salmonella;shigella; Yersinia enterocolitica; Yersinia pseudotuberculosis;Legionella pneumophila; Mycobacterium tuberculosis; Listeriamonocytogenes; Mycoplasma spp.; Pseudomonas fluorescens; Vibriocholerae; Haemophilus influenzae; Bacillus anthracis; Treponemapallidum; Leptospira; Borrelia; Corynebacterium diphtheriae;Francisella; Brucella melitensis; Campylobacter jejuni; Enterobacter;Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

In some embodiments, a membrane-receiver polypeptide complex may be usedto treat the infectious bacterial disease. A suitable receiverpolypeptide may comprise, for example, CD14 or a functional fragmentthereof. CD14 is associated with monocyte/macrophages and bindslipopolysaccharide associated with gram negative bacteria as well aslipoteichoic acid associated with the gram positive bacteria Bacillussubtilis. Other suitable receivers may comprise adenylate cyclase(Bordatella pertussis), Gal alpha 1-4Gal-containing isoreceptors (E.coli), glycoconjugate receptors (enteric bacteria), Lewis (b) bloodgroup antigen receptor (Heliobacter pylori), CR3 receptor, proteinkinase receptor, galactose N-acetylgalactosamine-inhibitable lectinreceptor, chemokine receptor (Legionella), annexin I (Leishmaniamexicana), ActA protein (Listeria monocytogenes), meningococcalvirulence associated Opa receptors (Meningococcus), acute over (α)5β3integrin (Mycobacterium avium-M), heparin sulphate proteoglycanreceptor, CD66 receptor, integrin receptor, membrane cofactor protein,CD46, GM1, GM2, GM3, and CD3 (Neisseria gonorrhoeae), KDEL receptor(Pseudomonas), epidermal growth factor receptor (Samonella typhiurium),β1 integrin (Shigella), nonglycosylated J774 receptor (Streptococci) orcombinations or functional fragments thereof.

In some embodiments, the synthetic membrane-receiver complex maycomprise more than one receiver. One receiver may function to interactwith the target, while the other receiver may modify the target, e.g.,disrupting the integrity of the target, marking the target fordegradation and/or inactivating the target. For example, if the targetis a bacterium, one receiver functions to interact with the targetbacterium (e.g., through an interaction with an epitope if the receivercomprises an antibody-like function). The other receiver may be capableof breaching the cell membrane of the bacterium. Suitable secondreceivers include, for example, lysozymes, bacteriocidal permeabilityincreasing peptides, proteases, and other pore forming antimicrobials.For example, a lysozyme receiver may hydrolyse 1,4-beta-linkages betweenN-acetylmuramic acid and N-acetyl-D-glucosamine residues in apeptidoglycan and between N-acetyl-D-glucosamine residues inchitodextrins of certain bacteria.

Alternatively, a second receiver may comprise a bacteriostatic orbactericidal agent that may be contacted with the bacterium. Yet anotheralternative is that the synthetic membrane-receiver complex comprises(e.g., through loading) a bacteriostatic or bactericidal agent that maybe contacted with the bacterium. Examples of bacteriostatic orbactericidal agents that may be associated with a receiver or thecomplex include, but are not limited to, beta-lactam compounds(penicillin, methicillin, nafcillin, oxacillin, cloxacillin,dicloxacilin, ampicillin, ticarcillin, amoxicillin, carbenicillin,piperacillin); cephalosporins & cephamycins (cefadroxil, cefazolin,cephalexin, cephalothin, cephapirin, cephradine, cefaclor, cefamandole,cefonicid, cefuroxime, cefprozil, loracarbef, ceforanide, cefoxitin,cefmetazole, cefotetan, cefoperazone, cefotaxime, ceftazidine,ceftizoxine, ceftriaxone, cefixime, cefpodoxime, proxetil, cefdinir,cefditoren, pivoxil, ceftibuten, moxalactam, cefepime); otherbeta-lactam drugs (aztreonam, clavulanic acid, sulbactam, tazobactam,ertapenem, imipenem, meropenem); cell wall membrane active agents(vancomycin, teicoplanin, daptomycin, fosfomycin, bacitracin,cycloserine); tetracyclines (tetracycline, chlortetracycline,oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline,tigecycline); macrolides (erythromycin, clarithromycin, azithromycin,telithromycin); clindamycin; choramphenicol; quinupristin-dalfopristin;linezolid; aminoglycosides (streptomycin, neomycin, kanamycin, amikacin,gentamicin, tobramycin, sisomicin, netilmicin); spectinomycin;sulfonamides (sulfacytine, sulfisoxazole, silfamethizole, sulfadiazine,sulfamethoxazole, sulfapyridine, sulfadoxine); trimethoprim;pyrimethamine; trimethoprim-sulfamethoxazole; fluoroquinolones(ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin); colistimethate sodium,methenamine hippurate, methenamine mandelate, metronidazole, mupirocin,nitrofurantoin, and polymyxin B. Examples of anti-mycobacteria drugsinclude, but are not limited to: isoniazid, rifampin, rifabutin,rifapentine, pyrazinamide, ethambutol, ethionamide, capreomycin,clofazimine, and dapsone.

In some embodiments, methods of treatment of bacterial infectiousdiseases are provided comprising co-administration of one or morebacteriostatic or bactericidal agents and the syntheticmembrane-receiver complex described herein, wherein co-administrationincludes administration of the bacteriostatic or bactericidal agentbefore, after or concurrent with administration of the syntheticmembrane-receiver complex.

In some embodiments, methods of treatment of bacterial infectiousdiseases are provided comprising administration of a pharmaceuticalcomposition comprising one or more bacteriostatic or bactericidal agentsand the synthetic membrane-receiver complex described herein.

In some embodiments, the receiver may sequester the target bacterium anddistribute it in the circulatory system without directly modifying thetarget. In certain embodiments, the synthetic membrane-receiver complexmay subject the associated target bacterium to degradation by thereticulo-endothelial system.

2. Fungal Infections

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for a fungal infection. Subjectssuffering from or at risk of developing a fungal infection may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In some embodiments, the target is a fungus. In certain embodiments, thetarget comprises a fungal antigen. In some embodiments, the fungalantigen comprises a cell surface antigen, a secreted toxin, or asecreted fungal antigen.

Fungemia (also known as candidemia, candedemia, and invasivecandidiasis) is the presence of fungi or yeasts in the blood. The mostcommonly known pathogen is Candida albicans, causing roughly 70% offungemias, followed by Candida glabrata with 10%, and Aspergillus with1%. Infections with T. glabrata, Candida tropicalis, C. krusei, and C.parapsilosis may also occur.

In some embodiments, a membrane-receiver polypeptide complex may be usedto treat the infectious fungal disease. In some embodiments, thesynthetic membrane-receiver complex may comprise more than one receiver.One receiver may function to interact with the target, while the otherreceiver may modify the target, e.g., disrupting the integrity of thetarget, marking the target for degradation and/or inactivating thetarget. The second receiver may comprise an antifungal agent that may becontacted with the fungus. In another embodiment, the syntheticmembrane-receiver complex comprises (e.g., through loading) anantifungal agent that may be contacted with the fungus.

Examples of antifungal agents that may be associated with a receiver orthe complex include, but are not limited to, allylamines; terbinafine;antimetabolites; flucytosine; azoles; fluconazole; itraconazole;ketoconazole; ravuconazole; posaconazole; voriconazole; glucan synthesisinhibitors; caspofungin; micafungin; anidulafungin; polyenes;amphotericin B; amphotericin B Lipid Complex (ABLC); amphotericin BColloidal Dispersion (ABCD); liposomal amphotericin B (L-AMB); liposomalnystatin; and griseofulvin.

In some embodiments, methods of treatment of fungal infectious diseasesare provided comprising co-administration of one or more antifungalagents and the synthetic membrane-receiver complex described herein,wherein co-administration includes administration of the antifungalagent before, after or concurrent with administration of the syntheticmembrane-receiver complex.

In some embodiments, methods of treatment of bacterial infectiousdiseases are provided comprising administration of a pharmaceuticalcomposition comprising one or more antifungal agents and the syntheticmembrane-receiver complex described herein.

In some embodiments, the receiver may sequester the target fungus anddistribute it in the circulatory system without directly modifying thetarget. In certain embodiments, the synthetic membrane-receiver complexmay subject the associated target fungus to degradation by thereticulo-endothelial system.

3. Parasite Infections

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for a parasitic infection.Subjects suffering from or at risk of developing a parasitic infectionmay be administered a pharmaceutical composition comprising thesynthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In some embodiments, the target is a parasite. In certain embodiments,the target comprises a parasitic antigen. In some embodiments, theparasitic antigen comprises a cell surface antigen, a secreted toxin, ora secreted parasitic antigen. Suitable targets include intestinal orblood-borne parasites, including protazoa.

Typically, blood-borne parasites are transmitted through an arthropodvector. Most important arthropod for transmitting parasitic infectionsare mosquitoes. Mosquitoes carry malaria and filarial nematodes. Bitingflies transmit African trypanosomiasis, leishmaniasis and several kindsof filariasis. Examples of parasites include, but are not limited to,trypanosomes; haemoprotozoa and parasites capable of causing malaria;enteric and systemic cestodes including taeniid cestodes; entericcoccidians; enteric flagellate protozoa; filarial nematodes;gastrointestinal and systemic nematodes and hookworms.

In some embodiments, a membrane-receiver polypeptide complex may be usedto treat the parasitic infection. In some embodiments, the syntheticmembrane-receiver complex may comprise more than one receiver. Onereceiver may function to interact with the target, while the otherreceiver may modify the target, e.g., disrupting the integrity of thetarget, marking the target for degradation and/or inactivating thetarget. The second receiver may comprise an anti-parasitic agent thatmay be contacted with the fungus. In another embodiment, the syntheticmembrane-receiver complex comprises (e.g., through loading) ananti-parasitic agent that may be contacted with the fungus.

Examples of anti-parasitic agents that may be associated with a receiveror the complex include, but are not limited to, antiprotozoal agents;eflornithine; furazolidone; melarsoprol; metronidazole; ornidazole;paromomycin sulfate; pentamidine; pyrimethamine; tinidazole;antimalarial agents; quinine; chloroquine; amodiaquine; pyrimethamine;sulphadoxine; proguanil; mefloquine; halofantrine; primaquine;artemesinin and derivatives thereof; doxycycline; clindamycin;benznidazole; nifurtimox; antihelminthics; albendazole;diethylcarbamazine; mebendazole; niclosamide; ivermectin; suramin;thiabendazole; pyrantel pamoate; levamisole; piperazine family;praziquantel; triclabendazole; octadepsipeptides; and emodepside.

In some embodiments, methods of treatment of parasitic infectiousdiseases are provided comprising co-administration of one or moreanti-parasitic agents and the synthetic membrane-receiver complexdescribed herein, wherein co-administration includes administration ofthe anti-parasitic agent before, after or concurrent with administrationof the synthetic membrane-receiver complex.

In some embodiments, methods of treatment of parasitic infectiousdiseases are provided comprising administration of a pharmaceuticalcomposition comprising one or more anti-parasitic agents and thesynthetic membrane-receiver complex described herein.

In some embodiments, the receiver may sequester the target parasite anddistribute it in the circulatory system without directly modifying thetarget. In certain embodiments, the synthetic membrane-receiver complexmay subject the associated target parasite to degradation by thereticulo-endothelial system.

4. Viral Infections

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for a viral infection. Subjectssuffering from or at risk of developing a viral infection may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In some embodiments, the target is a virus. In certain embodiments, thetarget comprises a viral antigen. In some embodiments, the viral antigencomprises an envelope antigen or a capsid antigen. Suitable viraltargets include adenovirus, coxsackievirus, hepatitis a virus,poliovirus, epstein-barr virus, herpes simplex, type 1, herpes simplex,type 2, human cytomegalovirus, human herpesvirus, type 8,varicella-zoster virus, hepatitis B virus, hepatitis C viruses, humanimmunodeficiency virus (HIV), influenza virus, measles virus, mumpsvirus, parainfluenza virus, respiratory syncytial virus, papillomavirus,rabies virus, and Rubella virus. Other suitable viral targets includeParamyxoviridae (e.g., pneumovirus, morbillivirus, metapneumovirus,respirovirus or rubulavirus), Adenoviridae (e.g., adenovirus),Arenaviridae (e.g., arenavirus such as lymphocytic choriomeningitisvirus), Arteriviridae (e.g., porcine respiratory and reproductivesyndrome virus or equine arteritis virus), Bunyaviridae (e.g.,phlebovirus or hantavirus), Caliciviridae (e.g., Norwalk virus),Coronaviridae (e.g., coronavirus or torovirus), Filoviridae (e.g.,Ebola-like viruses), Flaviviridae (e.g., hepacivirus or flavivirus),Herpesviridae (e.g., simplexvirus, varicellovirus, cytomegalovirus,roseolovirus, or lymphocryptovirus), Orthomyxoviridae (e.g., influenzavirus or thogotovirus), Parvoviridae (e.g., parvovirus), Picomaviridae(e.g., enterovirus or hepatovirus), Poxviridae (e.g., orthopoxvirus,avipoxvirus, or leporipoxvirus), Retroviridae (e.g., lentivirus orspumavirus), Reoviridae (e.g., rotavirus), Rhabdoviridae (e.g.,lyssavirus, novirhabdovirus, or vesiculovirus), and Togaviridae (e.g.,alphavirus or rubivirus). Specific examples of these viruses includehuman respiratory coronavirus, influenza viruses A-C, hepatitis virusesA to G, and herpes simplex viruses 1-9.

In some embodiments, a membrane-receiver polypeptide complex may be usedto treat the viral infection. In some embodiments, the syntheticmembrane-receiver complex may comprise more than one receiver. Onereceiver may function to interact with the target, while the otherreceiver may modify the target, e.g., disrupting the integrity of thetarget, marking the target for degradation and/or inactivating thetarget.

For example, if the target is a virus, one receiver functions tointeract with the target virus (e.g., through an interaction with aviral epitope if the receiver comprises an antibody-like function). Theother receiver may be capable of breaching the viral envelope or capsid.Suitable second receivers include, for example, antiviral agents,proteases, nucleases, antisense molecules, ribozymes, RNAi molecules(e.g., siRNA or shRNA), or other molecules that are toxic or detrimentalto the virus.

The second receiver may comprise an anti-viral agent that may becontacted with the virus. In another embodiment, the syntheticmembrane-receiver complex comprises (e.g., through loading) ananti-viral agent that may be contacted with the virus.

Examples of anti-viral agents that may be associated with a receiver orthe complex include, but are not limited to, thiosemicarbazones;metisazone; nucleosides and nucleotides; acyclovir; idoxuridine;vidarabine; ribavirin; ganciclovir; famciclovir; valaciclovir;cidofovir; penciclovir; valganciclovir; brivudine; ribavirin, cyclicamines; rimantadine; tromantadine; phosphonic acid derivatives;foscarnet; fosfonet; protease inhibitors; saquinavir; indinavir;ritonavir; nelfinavir; amprenavir; lopinavir; fosamprenavir; atazanavir;tipranavir; nucleoside and nucleotide reverse transcriptase inhibitors;zidovudine; didanosine; zalcitabine; stavudine; lamivudine; abacavir;tenofovir disoproxil; adefovir dipivoxil; emtricitabine; entecavir;non-nucleoside reverse transcriptase inhibitors; nevirapine;delavirdine; efavirenz; neuraminidase inhibitors; zanamivir;oseltamivir; moroxydine; inosine pranobex; pleconaril; and enfuvirtide.

In some embodiments, methods of treatment of viral infectious diseasesare provided comprising co-administration of one or more anti-viralagents and the synthetic membrane-receiver complex described herein,wherein co-administration includes administration of the anti-viralagent before, after or concurrent with administration of the syntheticmembrane-receiver complex.

In some embodiments, methods of treatment of viral infectious diseasesare provided comprising administration of a pharmaceutical compositioncomprising one or more antiviral agents and the syntheticmembrane-receiver complex described herein.

In some embodiments, the receiver may sequester the target virus anddistribute it in the circulatory system without directly modifying thetarget. In certain embodiments, the synthetic membrane-receiver complexmay subject the associated target virus to degradation by thereticulo-endothelial system.

Conditions Associated with Toxins and Poisons

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent toxicconditions or poisoning caused by toxins or poisons. Sepsis and septicshock, which represent major causes of mortality in modern intensivecare medicine, are caused by an inadequate inflammatory andimmunological host response to bacterial infection. Evidence suggeststhat the systemic spread of microbial toxins, rather than bacteremiaitself, is the crucial event in the pathogenesis. The endothelium is amain target of bacterial toxins. The resulting endothelial dysfunctionis believed to contribute to the underlying pathomechanisms and thecollapse of homeostasis of organ function.

Bacterial toxins targeting endothelial cells severely alter the behaviorof extravascular cells and circulating leukocytes via excessiveformation of vasoactive mediators and overexpression of adhesionmolecules (Grandel, Crit Rev Immunol, 2003).

Pore-forming toxins (PFTs) are one of the most common protein toxinsfound in nature. These toxins disrupt cells by forming pores in cellularmembranes and altering their permeability. In bacterial infections,attack by PFTs is a major virulence mechanism. It has been demonstratedthat the inhibition of the pore-forming a-toxin can reduce the severityof Staphylococcus aureus infections, and similar PFT-targeted strategieshave shown therapeutic potential against other pathogens, includingEscherichia coli, Listeria monocytogenes, Bacillus anthracis andStreptococcus pneumoniae. As well as their role in bacterialpathogenesis, PFTs are commonly used in venomous attacks by animals suchas sea anemones, scorpions and snakes. Over 80 families of PFTs havebeen identified, displaying diverse molecular structures and distinctiveepitopic targets (Zhang, Nature Nano, 2013).

A number of biomolecules show interactions with endotoxins, such aslipopolysaccharide-binding protein (LBP),bactericidal/permeability-increasing protein (BPI), amyloid P component,cationic protein, or the enzyme employed in the biological endotoxinassay (anti-LPS) factor from Limulus amebocyte lysate (LAL). Theseproteins are directly involved in the reaction of many different speciesupon administration of endotoxin.

In one embodiment, functional erythroid cells comprise a receiver thatcomprises an amino acid sequence derived from lipopolysaccharide bindingprotein (LBP). A population of functional erythroid cells comprising areceiver that comprises an amino acid sequence derived fromlipopolysaccharide binding protein (LBP) may be administered to asubject in need thereof in an amount effective to remove immunogeniclipopolysaccharide that may be in circulation as a result of a microbialinfection.

Further provided are methods of inducing toxin clearance. The methodsinclude administering to a subject in need thereof a population offunctional erythroid cells comprising a receiver that is capable ofinteracting with a toxin, such as e.g., an antibody, scFv or nanobodyreceiver, in an amount effective to clear toxins from circulation. Thecompositions comprising functional erythroid cells that comprise thetoxin-specific receiver may be administered to subjects that exhibitlevels of toxic metabolites or infectious agents such as anthrax,botulinum, cytokines, sarin, hemolysin, venoms, and including, but notlimited to, those in table 5.

In one embodiment, functional erythroid cells comprise a receiver thatcomprises an amino acid sequence derived from the endotoxin receptorCD14. A population of functional erythroid cells comprising a receiverthat comprises an amino acid sequence derived from the endotoxinreceptor CD14 may be administered to a subject in need thereof in anamount effective to bind to a target endotoxin in circulation. Suchmethods may be employed to sequester the toxin and reduce the amount oftissue damage that would otherwise occur within the vasculature anddissipating its pathogenic effects in a less acute manner.

In one embodiment, the receiver interacts with cell-free circulatingDNA. In one embodiment, the functional erythroid cell expressesexogenous gene encoding a receiver comprising an amino acid sequencederived from a DNA-interacting polypeptide, such as, e.g., DNase, atranscription factor DNA binding domain or histone fragments. The DNase,DNA binding domain or histone fragment may be expressed as a fusionprotein. In other embodiments, the DNAse, DNA binding domain, histonefragment or another receiver with affinity to circulating DNA is loadedinto or onto the erythroid cell. In one embodiment, the receiver is aDNase, DNA binding domain or histone fragment that is localizedextracellularly.

A hallmark of apoptosis is DNA degradation, in which chromosomal DNA isfirst cleaved into large fragments (50-300 kb) and subsequently intomultiples of nucleosomal units (180-200 bp) (Nagata, Cell Death Differ,2003). The contents of apoptotic cells are ingested by phagocytes orneighboring cells and the DNA is completely digested by DNase II inlysosomes (Nagata, Cell Death Differ, 2003). Thus, DNA fragmentsreleased by apoptosis may be removed before appearing in thecirculation. In instances where the engulfment of apoptotic bodies isimpaired or cell death is increased an inflammatory response may occur.For example, autoimmunity occurs frequently in cancer and otherconditions involving increased circulating DNA (Viorritto, Clin Immunol,2007).

Extracellular DNA, or circulating cell free DNA (cf-DNA), is present inblood plasma. These cf-DNAs, at least part of them, are believed tooriginate from cancer cells and contain a number of cancer specificentities, including oncogenes, tumor suppressor genes, aberrantmicrosatellites, aberrant DNA methylation genes, and rearrangedchromosomal DNA. The term, ‘genometastasis’ has been proposed todescribe the phenomena of an apoptotic body containing DNA thathorizontally enters and transforms healthy cells (Garcia-Olmo, ExpertOpinion on Bio Therapy, 2012).

In certain embodiments, functional erythroid cells comprising a receiverspecific for circulating DNA are administered to a subject in needthereof in an amount effective to treat a DNA-driven pathogenesis, suchas systemic lupus erythematosus and cancers suspected of genometastasis.In some embodiments, functional erythroid cells comprise anextracellular receiver comprising DNAse fused to the N terminal ofglycophorin A such that it is capable of degrading circulating DNAwithin the vasculature.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target toxin or poison in a subject(e.g., a human) suffering from or at risk of developing a toxiccondition or poisoning. The methods include administering apharmaceutical composition comprising a synthetic membrane-receiverpolypeptide complex described herein. The pharmaceutical composition isadministered in an amount effective to substantially reduce thecirculatory concentration of the target toxin or poison. In certainembodiments, the administration is carried out intravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds, sequesters, and/or degrades a toxin or poison, such as apathogenic toxin, a venom, a prion protein, a cytokine, a metal (e.g.,heavy metal), or an alcohol (e.g., methanol) that is present in thecirculatory system of the subject. Conditions associated with toxins orpoisons include, but are not limited to bacterial toxin-induced shock,spider venom-induced shock, prion diseases, cytokine storm, ironpoisoning, copper poisoning, Wilson disease, heavy metal poisoning,methanol poisoning and those listed in table 6 and table 8.

Further provided are methods of inducing toxin clearance. In specificembodiments, the methods include administering to a subject in needthereof a pharmaceutical composition of erythrocyte cells comprising areceiver provided herein in an amount sufficient to induce toxinclearance in the subject. The compositions may be administered tosubjects that exhibit levels of toxic metabolites or infectious agentssuch as anthrax, botulinum, cytokines, sarin, hemolysin, venoms, andthose included, but not limited to table 5.

In one embodiment the disease or condition is alpha hemolysin poisoning,the receiver is an antibody-like binder to alpha hemolysin or fragmentthereof, and the target is alpha hemolysin.

In one embodiment the disease or condition is antrax toxin poisoning,the receiver is an antibody-like binder to anthrax toxin or fragmentthereof, and the target is anthrax toxin.

In one embodiment the disease or condition is bacterial toxin-inducedshock, the receiver is an antibody-like binder to bacterial toxin orfragment thereof, and the target is bacterial toxin.

In one embodiment the disease or condition is botulinum toxin poisoning,the receiver is an antibody-like binder to botulinum toxin or fragmentthereof, and the target is botulinum toxin.

In one embodiment the disease or condition is prion disease caused byPRP, the receiver is an antibody-like binder to prion protein PRP orfragment thereof, and the target is prion protein PRP.

In one embodiment the disease or condition is prion disease caused byPRPc, the receiver is an antibody-like binder to prion protein PRPc orfragment thereof, and the target is prion protein PRPc.

In one embodiment the disease or condition is prion disease caused byPRPsc, the receiver is an antibody-like binder to prion protein PRPsc orfragment thereof, and the target is prion protein PRPsc.

In one embodiment the disease or condition is prion disease caused byPRPres, the receiver is an antibody-like binder to prion protein PRPresor fragment thereof, and the target is prion protein PRPres.

In one embodiment the disease or condition is sepsis or cytokine storm,the receiver is an antibody-like binder to cytokines or duffy antigenreceptor of chemokines (DARC) or fragment thereof, and the target iscytokines.

Wilson's disease is caused by a failure of copper metabolism and abuildup of copper in liver, brain, and other organs. Copper chelatorsare used clinically, for example D-penicillamine, but they suffer fromshort half-lives that reduce their therapeutic efficacy. In oneembodiment, the receiver on the surface of a synthetic membrane-receivercomplex is D-penicillamine Administration of the syntheticmembrane-receiver complex will allow D-penicillamine to remain incirculation for substantially longer than free D-penicillamine, thuscapturing copper for a longer period of time and providing a clinicalbenefit in Wilson's disease.

In one embodiment the disease or condition is spider venom poisoning,the receiver is an antibody-like binder to spider venom or fragmentthereof, and the target is spider venom.

1. Toxins

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for botulinum toxin (BTX)poisoning. Subjects suffering from or at risk of developing BTXpoisoning may be administered a pharmaceutical composition comprisingthe synthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising an antibody domain or antibody-likedomain that binds to BTX of any of the types A-H, or a derivative orfunctional fragment thereof. A suitable receiver may be exhibited on thesurface of the synthetic membrane-receiver polypeptide complex. Thesuitable receiver is capable of binding to BTX and preventing BTX fromcarrying out its function.

BTX is produced by Clostridium botulinum and is a potent neurotoxin withan estimated human lethal dose of 1.3-2.1 ng/kg intravenously (Arnon etal. 2001 J Am Med Assoc 285(8):1059). BTX is a protease that attacks oneof the fusion proteins (SNAP-25, syntaxin or synaptobrevin) at aneuromuscular junction, preventing vesicles from anchoring to themembrane to release acetylcholine. By inhibiting acetylcholine release,the toxin interferes with nerve impulses and causes flaccid (sagging)paralysis of muscles.

2. Prions—Creutzfeldt-Jakob Disease

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for Creutzfeldt-Jakob Disease(CJD) caused by prion protein in the scrapie form (PrPsc). Subjectssuffering from or at risk of developing CJD may be administered apharmaceutical composition comprising the synthetic membrane-receiverpolypeptide complex described herein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising an antibody domain or antibody-likedomain that binds to PrPsc or a derivative or functional fragmentthereof. A suitable receiver may be exhibited on the surface of thesynthetic membrane-receiver polypeptide complex. The suitable receiveris capable of binding to PrPsc and preventing PrPsc from carrying outits function.

PrPsc is a misfolded form of PrP that can induce normal PrP to misfoldin an autocatalytic fashion. PrPsc is protease resistant and formsinsoluble aggregates and fibrils that damage cells. In CJD, the PrPscaggregates and fibrils lead to rapid neurodegeneration, causing thebrain tissue to develop holes and take on a sponge-like texture.

3. Cytokines

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for sepsis. Subjects sufferingfrom or at risk of developing sepsis poisoning may be administered apharmaceutical composition comprising the synthetic membrane-receiverpolypeptide complex described herein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises one or more receivers comprising an antibody domain,antibody-like domain, or cytokine receptor domain that bind to one ormore of the cytokines tumor necrosis factor alpha (TNFa), interferongamma (IFNg), or interleukin-2 (IL-2) or a derivative or functionalfragment thereof. A suitable receiver may be exhibited on the surface ofthe synthetic membrane-receiver polypeptide complex. The suitablereceiver is capable of binding to the cytokine and preventing thecytokine from carrying out its function, e.g., by preventing thecytokine from biding to its native receptor.

Cytokines like TNFa, IFNg, and IL-2 are produced by immune cells inresponse to infection and are powerful inflammatory stimuli for otherimmune cells. In sepsis, a serious bacterial infection induceswhole-body inflammation driven by a storm of cytokines, which triggersmulti-organ failure, acute respiratory distress, heart failure,encephalopathy, and edema.

Diseases and Conditions Associated with the Accumulation of Lipids orCholesterols

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases andconditions associated with the accumulation of lipids or cholesterols.

In one embodiment, the receiver interacts with one or more lipids. Inone embodiment, the functional erythroid cell expresses a exogenous geneencoding an amino acid sequence derived from a lipase. The lipase may beexpressed as a full-length protein or a fragment thereof. The lipase maybe expressed as a fusion protein. In other embodiments, the lipaseprotein receiver or another receiver with affinity to lipids is loadedinto or onto the erythroid cell. The lipase protein receiver or theother receiver with affinity to lipids may be localized intracellularlyor extracellularly. In one embodiment, the receiver comprises an aminoacid sequence derived from lipoprotein lipase.

Hyperlipidemia or hyperlipoproteinemia is an excess of lipids, largelycholesterol and triglycerides, in the blood. Lipids travel in the bloodattached to proteins to remain dissolved while in circulation.Hyperlipidemia, in general, can be divided into two subcategories;hypercholesterolemia, in which there is a high level of cholesterol andhypertriglyceridemia, in which there is a high level of triglycerides,the most common form of fat. Excess LDL cholesterol contributes to theblockage of arteries, which eventually leads to heart attack. Populationstudies have shown that the higher the level of LDL cholesterol, thegreater the risk of heart disease.

Hyperlipidemia usually has no noticeable symptoms and tends to bediscovered during routine examination or evaluation for atheroscleroticcardiovascular disease. However, deposits of cholesterol (known asxanthomas) may form under the skin (especially around the eyes or alongthe Achilles tendon) in individuals with familial forms of the disorderor in those with very high levels of cholesterol in the blood.Individuals with hypertriglyceridemia may develop numerous pimple-likelesions across their body. Extremely high levels of triglycerides mayalso result in pancreatitis, a severe inflammation of the pancreas thatmay be life-threatening.

In certain embodiments, functional erythroid cells comprise a receiverthat is capable of interacting with a lipid, or has affinity to a targetlipid or target lipid-associated molecule listed in table 5. In certainembodiments, a population of functional erythroid cells comprising areceiver that is capable of interacting with a lipid or comprising areceiver that comprises an amino acid sequence derived from lipoproteinlipase is administered to a subject in need thereof in an amounteffective to treat or prevent hyperlipidemia.

In certain embodiments, a population of functional erythroid cellscomprising a receiver that is capable of interacting with a lipid orcomprising a receiver that comprises an amino acid sequence derived fromlipoprotein lipase is administered to a subject in need thereof in anamount effective to remove chylomicrons, which are lipoprotein particlesconsisting of lipids, protein, and cholesterol, from the bloodcirculation. In some embodiments, the receiver is lipoprotein lipase andthe receiver is localized on the surface of the erythroid cell. Incertain embodiments, a population of functional erythroid cellscomprising a receiver that comprises an amino acid sequence derived fromlipoprotein lipase is administered to a subject in need thereof in anamount effective to treat, alleviate or prevent lipoprotein lipasedeficiency. Familial lipoprotein lipase deficiency is a group of raregenetic disorders in which a person lacks the ability to break downlipids, which causes a large amount of fat to build up in the blood.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target lipid or cholesterol in a subject(e.g., a human) suffering from or at risk of developing a disease orcondition associated with the accumulation of lipids or cholesterols.The methods include administering a pharmaceutical compositioncomprising a synthetic membrane-receiver polypeptide complex describedherein. The pharmaceutical composition is administered in an amounteffective to substantially reduce the circulatory concentration of thetarget lipid or cholesterol. In certain embodiments, the administrationis carried out intravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds, sequesters, and/or degrades a target lipid or cholesterol, or acomplex or aggregate that comprises a lipid or cholesterol, that ispresent in the circulatory system of the subject. Reduction in theamount or concentration of circulating lipids or cholesterols andassociated complexes therewith may reduce or alleviate cardiovascularand other circulatory problems. Diseases or conditions associated withthe accumulation of lipids or cholesterols include, but are not limitedto lipoprotein lipase deficiency, hypercholesterolemia, coronary arterydisease and those listed in table 6 and table 8.

In one embodiment the disease or condition is hypercholesterolemia, thereceiver is an antibody-like binder to low-density lipoprotein (LDL),LDL receptor or fragment thereof, and the target is LDL.

In one embodiment the disease or condition is hypercholesterolemia, thereceiver is an antibody-like binder to high-density lipoprotein (HDL) orHDL receptor or fragment thereof, and the target is HDL.

In one embodiment the disease or condition is lipoprotein lipasedeficiency, the receiver is lipoprotein lipase or fragment thereof, andthe target is chilomicrons and very low density lipoproteins (VLDL).

Lipoprotein Lipase Deficiency (Glybera)

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for lipoprotein lipasedeficiency. Subjects suffering from or at risk of developing lipoproteinlipase deficiency may be administered a pharmaceutical compositioncomprising the synthetic membrane-receiver polypeptide complex describedherein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising the enzyme lipoprotein lipase or aderivative or functional fragment thereof. A suitable receiver may beexhibited on the surface of the synthetic membrane-receiver polypeptidecomplex. The suitable receiver is capable of hydrolyzing triglyceridesin lipoproteins, such as those found in chylomicrons and verylow-density lipoproteins (VLDL), into two free fatty acids and onemonoacylglycerol molecule.

Lipoprotein lipase deficiency is a rare disorder in which afflictedindividuals lack the ability to produce lipoprotein lipase enzymesnecessary for effective breakdown of fatty acids. The disorder usuallypresents in childhood and is characterized by very severehypertriglyceridemia with episodes of abdominal pain, recurrent acutepancreatitis, eruptive cutaneous xanthomata, and hepatosplenomegaly.Clearance of chylomicrons from the plasma is impaired, causingtriglycerides to accumulate in plasma and the plasma to have a milkyappearance. Symptoms usually resolve with restriction of total dietaryfat to ≦20 grams/day

Diseases and Conditions Associated with Infected, Aberrant or OncogenicCells

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases andconditions associated with infected, aberrant or oncogenic cells, suchas, e.g., cancer.

In one embodiment, the receiver interacts with a cancer stem cell (CSC)or another cancer-associated circulatory cell. In one embodiment, thefunctional erythroid cell expresses a exogenous gene encoding anantibody, scFv or nanobody specific for a CSC antigen. The antibody,scFv or nanobody may be expressed as a fusion protein. In otherembodiments, the antibody, scFv or nanobody receiver or another receiverwith affinity to circulating cancer cells is loaded into or onto theerythroid cell. In one embodiment, the receiver is an antibody, scFv ornanobody that is localized extracellularly. In certain embodiments, theantibody, scFv or nanobody receiver is specific for a CSC antigenselected from CD44, CD47, and MET.

Cancer stem cells (CSCs), which comprise a small fraction of cancercells, are believed to constitute the origin of most human tumors. Oneof the key steps in the metastatic cascade is the migration of tumorcells away from the primary tumor, and CSCs are likely associated withthis migration. Most adult tissues maintain some aspect of migratorycapacity through the ability to generate an epithelial to mesenchymaltransition (EMT)-like process during wound healing, tissue regenerationand organ fibrosis. It has been hypothesized that CSCs may also activatetheir migration through the process of EMT.

A number of studies have linked circulating tumor cells (CTCs) to tumorprogression in a variety of solid tumors, and CTC enumeration has begunto be utilized as a prognostic tool in patients with metastatic breast(Cristofanilli et al., 2004), colon (Cohen et al., 2008) and prostatecancer (Danila et al., 2007). Potentially, a fraction of CTCs have CSCactivity, and it is hypothesized that CSCs in a primary tumor whichenter the circulation become circulating CSCs and remain so until theylodge or home to a target organ. CTCs isolated from patients withmelanomas have been found to generate metastases in xenotransplantationmodels (Ma et al., 2010, Shiozawa, Pharm and Thera, 2013).

The vasculature is a powerful conduit for the proliferation of variouscirculating tumor cells, metastases, and cancer stem cells. In certainembodiments, functional erythroid cells comprising a receiver specificfor circulating cancer cells are administered to a subject in needthereof in an amount effective to treat or prevent metastases. Incertain embodiments, populations of functional erythroid cellscomprising a receiver specific for circulating cancer cells areadministered to a subject in need thereof in an amount effective tointeract with CSCs or CTCs to clear them from circulation, e.g., byfacilitating degradation in the liver. In some embodiments, functionalerythroid cells comprise an antibody, scFv, or nanobody receiverspecific for CD44, CD47, or MET (three characteristic surface antigensof CTC). Suitable cancer cells that may be cleared by the erythroidcells described herein include, but are not limited to, cells associatedwith the cancers listed in table 5 and table 8.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target cell in a subject (e.g., a human)suffering from or at risk of developing a disease or conditionassociated with an infected, aberrant or oncogenic cell. The methodsinclude administering a pharmaceutical composition comprising asynthetic membrane-receiver polypeptide complex described herein. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the target cell.In certain embodiments, the administration is carried out intravenously.

In certain embodiments, synthetic membrane-receiver polypeptidecomplexes are administered that comprise a receiver that specificallybinds, sequesters, and/or degrades a target cell, such as an infected,aberrant or oncogenic cell that is present in the circulatory system ofthe subject. Reduction in the amount or concentration of circulatingtarget cells may reduce or alleviate conditions associated with theinfected, aberrant or oncogenic cell, such as, e.g., an infection orcancer. Diseases or conditions associated with infected, aberrant oroncogenic cells include, but are not limited to cancer and those listedin table 6 and table 8.

In one embodiment the disease or condition is cancer, the receiver is anantibody-like binder to CD44 or fragment thereof, and the target is acirculating tumor cell.

In one embodiment the disease or condition is cancer, the receiver is anantibody-like binder to EpCam or fragment thereof, and the target is acirculating tumor cell.

In one embodiment the disease or condition is cancer, the receiver is anantibody-like binder to Her2 or fragment thereof, and the target is acirculating tumor cell.

In one embodiment the disease or condition is cancer, the receiver is anantibody-like binder to EGFR or fragment thereof, and the target is acirculating tumor cell.

In one embodiment the disease or condition is cancer (B cell), thereceiver is an antibody-like binder to CD20 or fragment thereof, and thetarget is a cancerous B cell.

In one embodiment the disease or condition is cancer (B cell), thereceiver is an antibody-like binder to CD19 or fragment thereof, and thetarget is a cancerous B cell.

Circulating Cancer Cell

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for cancer. Subjects sufferingfrom or at risk of developing cancer may be administered apharmaceutical composition comprising the synthetic membrane-receiverpolypeptide complex described herein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising an antibody domain or antibody-likedomain that binds to a circulating cancer cell, e.g., a proliferative Bcell, via a cancer cell specific receptor, e.g., CD19, or a derivativeor functional fragment thereof. A suitable receiver may be exhibited onthe surface of the synthetic membrane-receiver polypeptide complex. Thesuitable receiver is capable of binding to CD19 on the circulatingcancer cell and promoting the clearance of the CD19-expressing cancercell.

CD19 is a common receptor to B cells, and is a validated marker for Bcell cancers including B cell leukemias and lymphomas (Scheuermann andRacila, (1995) Leukemia and Lymphoma 18 (5): 385-397. It is increasinglyunderstood to play an additional role in the proliferation of B cells incancer by stabilizing the Myc oncoprotein (Chung et al. 2012, J ClinInvest 122(6):2257). In B cell cancers, proliferative B cells overwhelmlymph nodes and bone marrow. Strategies to target and clear these Bcells, including antibody therapy (Rituximab), are accepted as part ofthe standard of care.

Tumor metastasis is the main driver of cancer mortality and therapiestargeting metastasis are limited in number, mechanism of action andefficacy. Hematogenous tumor cell spreading (via bloodstream) is acommon route for many carcinomas and is a highly complex processinvolving primary site detachment, migration, transport into thebloodstream, tumor cell adhesion in the vasculature and proliferation atthe metastatic site.

Diseases and Conditions Associated with a Metabolic Defect

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases andconditions associated with a metabolic defect. A schematic example of asynthetic membrane-receiver polypeptide complex is shown in FIG. 13A.

As described herein, many small molecule metabolites can diffuse acrossthe membrane of, e.g., erythroid cells comprising a suitable receiver,or are actively transported by defined transmembrane channels (see,e.g., Tunnicliff, Comp. Biochem. Physiol. 1994). Some metabolites,however, may require additional assistance to reach the intracellularlylocalized receiver enzyme, thus the synthetic membrane-receiver complexmay optionally comprise a transporter.

In one embodiment, the surface exposed receiver polypeptide may shuttlethe substrate across the cell membrane into the syntheticmembrane-receiver complex, e.g., an erythroid cell comprising areceiver. The functional erythroid cell comprising a receiver maycontain multiple receiver polypeptides, including, but not limited to,the receiver polypeptides listed in Table 7. The receiver polypeptidesmay increase the cell's capabilities to transport metabolites or othersubstrates across the membrane. For example, a Glut1 transporter may becontained in the functional erythroid cell's membrane in combinationwith an intracellularly expressed receiver glucokinase, such that theerythroid cell internalizes and phosphorylates an amount of glucosegreater than that of a non-modified erythroid cell. Erythroid cellscomprising a receiver glucokinase may be used to reduce blood glucoselevels. Diabetes mellitus type II is associated with hyperglycemia as aresult of insulin resistance and relative lack of insulin. Thehyperglycemia may be alleviated by erythroid cells comprising a receiverglucokinase that capture glucose through surface-localized, receiverGlut1 and phosphorylation by an intracellularly localized, receiverglucokinase. Modified glucose may be unable to exit the cell. Thesynthetic membrane-receiver complex acts as a “buffer” to respond tohyperglycemic conditions.

Optionally, a second receiver polypeptide may be present in thefunctional erythroid cell that exhibits increase transport capabilities.The second receiver polypeptide may be localized intracellularly. Thesecond intracellularly localized receiver polypeptide can enzymaticallymodify, convert, change or otherwise alter the target substrate that wasshuttled into the cell by the first receiver polypeptide localized onthe cell surface.

In specific embodiments, methods are provided for modulating thecirculatory concentration of a target metabolite in a subject (e.g., ahuman) suffering from or at risk of developing a disease or conditionassociated with a metabolic defect. The methods include administering apharmaceutical composition comprising a synthetic membrane-receiverpolypeptide complex described herein. The pharmaceutical composition isadministered in an amount effective to substantially modulate thecirculatory concentration of the target metabolite. In some embodiments,the target metabolite is present or present in elevated levels incirculation and the amount or concentration of the target metabolite isreduced. For example if the level or concentration of a metabolite istoxic, the toxic target metabolite may be degraded or the toxic targetmetabolite may be converted into another non-toxic product (e.g., bycatalytic action of the receiver). In some embodiments, a non-targetmetabolite is absent or present in depressed levels in circulation and atarget metabolite is converted to the non-target metabolite so that itslevel or concentration is increased. In such embodiments, the absence ofdepressed levels of the non-target metabolite is associated with themetabolic disease or disorder and conversion of the target metabolite tothe non-target metabolite can at least partially restore or replenishthe level or concentration of the non-target metabolite, therebytreating or preventing the metabolic disease. In certain embodiments,the administration is carried out intravenously. Diseases or conditionsassociated with a metabolic defect include, but are not limited tomitochondrial neurogastrointestinal encephalomyopathy (MNGIE), adenosinedeaminase (ADA) deficiency, purine nucleoside phosphorylase (PNP)deficiency, phenylketonuria, alkaptonuria, homocystinuria, primaryhyperoxaluria and those listed in table 6 and table 8.

In specific embodiments, methods of treating a metabolic disease includeadministering to a subject in need thereof a pharmaceutical compositionof erythrocyte cells comprising a receiver provided herein in an amountsufficient to treat the metabolic disease. The compositions may beadministered to subjects that exhibit disorders of carbohydratemetabolism, amino acid metabolism, organic acid metabolism,mitochondrial metabolism, fatty acid metabolism, purine-pyrimidinemetabolism, steroid metabolism, peroxisomal function, lysosomal storage,or urea cycle. Of these disorders, specific indications includeADA-SCID, primary hyperoxaluria, and phenylketonuria, as well as, butnot limited to, the conditions listed in table 6 and table 8.

In one embodiment the disease or condition is 3-methylcrotonyl-CoAcarboxylase deficiency, the receiver is 3-methylcrotonyl-CoA carboxylaseor fragment thereof, and the target is 3-hydroxyvalerylcarnitine,3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA).

In one embodiment the disease or condition is acute intermittentporphyria, the receiver is porphobilinogen deaminase or fragmentthereof, and the target is porphobilinogen.

In one embodiment the disease or condition is adeninephosphoribosyltransferase deficiency, the receiver is adeninephosphoribosyltransferase or fragment thereof, and the target isinsoluble purine 2,8-dihydroxyadenine.

In one embodiment the disease or condition is adenosine deaminasedeficiency, the receiver is adenosine deaminase or fragment thereof, andthe target is adenosine.

In one embodiment the disease or condition is alkaptonuria, the receiveris homogentisate oxidase or fragment thereof, and the target ishomogentisate.

In one embodiment the disease or condition is argininemia, the receiveris ammonia monooxygenase or fragment thereof, and the target is ammonia.

In one embodiment the disease or condition is argininosuccinateaciduria, the receiver is ammonia monooxygenase or fragment thereof, andthe target is ammonia.

In one embodiment the disease or condition is citrullinemia type I, thereceiver is ammonia monooxygenase or fragment thereof, and the target isammonia.

In one embodiment the disease or condition is citrullinemia type II, thereceiver is ammonia monooxygenase or fragment thereof, and the target isammonia.

In one embodiment the disease or condition is glutaric acidemia type I,the receiver is lysine oxidase or fragment thereof, and the target is3-hydroxyglutaric and glutaric acid (C5-DC) amd lysine.

In one embodiment the disease or condition is gout with hyperuricemia,the receiver is uricase or fragment thereof, and the target is uric acid(urate crystals).

In one embodiment the disease or condition is hemolytic anemia due topyrimidine 5′ nucleotidase deficiency, the receiver is pyrimidine 5′nucleotidase or fragment thereof, and the target is pyrimidines.

In one embodiment the disease or condition is homocystinuria, thereceiver is Cystathionine B synthase or fragment thereof, and the targetis homocysteine.

In one embodiment the disease or condition ishyperammonemia/ornithinemia/citrullinemia (ornithine transporterdefect), the receiver is ammonia monooxygenase or fragment thereof, andthe target is ammonia.

In one embodiment the disease or condition is isovaleric acidemia, thereceiver is leucine metabolizing enzyme or fragment thereof, and thetarget is leucine.

In one embodiment the disease or condition is Lesch-Nyhan syndrome, thereceiver is uricase or fragment thereof, and the target is uric acid.

In one embodiment the disease or condition is maple syrup urine disease,the receiver is a leucine metabolizing enzyme or fragment thereof, andthe target is leucine.

In one embodiment the disease or condition is methylmalonic acidemia(vitamin b12 non-responsive), the receiver is methylmalonyl-CoA mutaseor fragment thereof, and the target is methylmalonate.

In one embodiment the disease or condition is mitochondrialneurogastrointestinal encephalomyopathy (MNGIE), the receiver isthymidine phosphorylase or fragment thereof, and the target isthymidine.

In one embodiment the disease or condition is phenylketonuria, thereceiver is phenylalanine hydroxylase, phenylalanine ammonia lyase orfragment thereof, and the target is phenylalanine.

In one embodiment the disease or condition is primary hyperoxaluria, thereceiver is oxalate oxidase or fragment thereof, and the target isoxalate.

In one embodiment the disease or condition is propionic acidemia, thereceiver is a propionate convertase or fragment thereof, and the targetis proprionyl coA.

In one embodiment the disease or condition is purine nucleosidephosphorylase deficiency, the receiver is purine nucleosidephosphorylase or fragment thereof, and the target is Inosine and/ordGTP.

In one embodiment the disease or condition is transferase deficientgalactosemia (galactosemia type 1), the receiver is galactosedehydrogenase or fragment thereof, and the target isgalactose-1-phosphate.

In one embodiment the disease or condition is tyrosinemia type 1, thereceiver is tyrosine phenol-lyase or fragment thereof, and the target istyrosine.

1. Adenosine Deaminase (ADA) Deficiency

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for adenosine deaminase (ADA)deficiency. Subjects suffering from or at risk of developing ADAdeficiency may be administered a pharmaceutical composition comprisingthe synthetic membrane-receiver polypeptide complex described herein totreat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising adenosine deaminase (ADA) or aderivative or functional fragment thereof. A suitable receiver may beexhibited on the surface or on the unexposed side of the syntheticmembrane-receiver polypeptide complex and may be administered to convertdeoxy-adenosine to deoxy-inosine, thereby preventing the build-up oftoxic deoxy-adenosine levels.

In certain embodiments, compositions comprising a plurality offunctional erythroid cells comprising an adenosine deaminase (ADA)receiver are provided. Such compositions may be used to treat subjectsthat exhibit ADA-severe combined immunodeficiency (SCID).

Subjects that exhibit an ADA-deficiency are experiencing a build-up ofdeoxy-adenosine in the body's tissues. The high deoxy-adenosine levelsare toxic to immature leukocytes. As a consequence, the subject'sadaptive immune response is impaired, which makes them highlysusceptible to infection. ADA is an endogenous enzyme produced by a widevariety of cells, including erythrocytes. ADA is responsible forconverting deoxy-adenosine to deoxy-inosine, thereby preventing thebuild-up of toxic deoxy-adenosine levels. Available enzyme replacementtherapies source ADA from bovine intestine. The foreign-sourced ADA issubject to immunogenic reactions and inhibitor development. Inhibitordevelopment may occur when a subject's immune system develops theability to clear and/or alter a therapeutic molecule such that itstherapeutic effect is decreased. In addition, the emergence of newvariant Creutzfeldt-Jakob disease has raised concerns about sourcing ADAfrom bovine intestine (Booth 2009, Biologics: Targets and Therapy).

In certain embodiments, provided herein are compositions comprising aplurality of functional erythroid cells comprising an adenosinedeaminase (ADA) receiver which may be administered to ADA-SCID subjectsto elevate the level of ADA over that of the endogenous levels ofexisting wild type cells in the ADA-SCID subject. Most ADA-SCID subjectsseverely lack a functioning deoxy-adenosine metabolism. The erythroidcells may contain exogenous ADA within their intracellular space. Theintracellularly localized exogenous ADA receiver polypeptide may thenconvert deoxy-adenosine to deoxy-inosine, thereby lowering the levels ofdeoxy-adenosine. Deoxy-adenosine crosses the cell membrane, is convertedto deoxy-inosine, and diffuses back into circulation. This may besufficient to preserve immature leukocyte populations, thereby treatingthe disease. In some embodiments, the adenosine deaminase receiver isexpressed as a fusion to the C terminus of hemoglobin beta such that theADA is retained in the functional erythroid cell during enucleation.Alternatively, the ADA gene is fused to the part of the gene encodingthe C terminus of glycophorin A such that upon expression it is tetheredto the intracellular portion of the transmembrane antigen.

2. Phenylketonuria (PKU)

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for phenylketonuria (PKU).Subjects suffering from or at risk of developing PKU may be administereda pharmaceutical composition comprising the synthetic membrane-receiverpolypeptide complex described herein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising phenylalanine ammonia lyase (PAL) or aderivative or functional fragment thereof.

In another embodiment, the synthetic membrane-receiver polypeptidecomplex comprises a receiver comprising phenylalanine hydroxylase (PAH)or a derivative or functional fragment thereof.

A suitable receiver may be exhibited on the surface or on the unexposedside of the synthetic membrane-receiver polypeptide complex and may beadministered to convert phenylalanine to tyrosine, thereby preventingthe build-up of toxic phenylalanine levels to treat or prevent PKU.

In specific embodiments, compositions comprising a plurality offunctional erythroid cells comprising a phenylalanine ammonia lyase(PAL) receiver are provided. Such compositions may be used to treatsubjects that exhibit or are diagnosed with phenylketonuria (PKU).

Subjects diagnosed with PKU are deficient in phenylalanine ammoniahydroxylase (PAH) activity due to an enzyme mutation or productiondeficiency. PAH, along with its cofactor tetrahydrobiopterin, isresponsible for converting phenylalanine to tyrosine. PAH deficiencyleads to phenylalanine accumulation and is associated with severalneurological disorders.

PAL is an enzyme isolated from plants, yeast, and fungi chrysanthemi.PAL is a large, 270 kDa enzyme that can elicit a strong immunogenicreaction. It is also quickly cleared from the body, therefore requiringlarge, frequent infusions. Even in its pegylated form, PAL only remainsin circulation for approximately three days. The short half-life makesPAL treatment difficult for patients to adhere to (Gamez, MolecularTherapy 2005).

In certain embodiments, provided herein are compositions comprising aplurality of functional erythroid cells comprising a phenylalanineammonia lyase (PAL) receiver, which may be administered tophenylketonuria (PKU) subjects to treat phenylalanine accumulation. Thefunctional erythroid cells may contain exogenous PAL within theirintracellular space. The intracellularly localized exogenous PALpolypeptide may then convert phenylalanine to trans-cinnamic acid, abenign metabolite, thereby lowering the levels of phenylalanine.Phenylalanine crosses the cell membrane, is converted to trans-cinnamicacid, and diffuses back into circulation. This may be sufficient toreduce phenylalanine concentrations in the blood.

In specific embodiments, compositions comprising a plurality offunctional erythroid cells comprising a phenylalanine hydroxylase (PAH)receiver are provided. Such compositions may be used to treat subjectsthat exhibit or are diagnosed with phenylketonuria (PKU). PAH is anenzyme that can be isolated from bacteria or mammals. PAH fromChromobacterium violaceum is a monomeric ˜30 kDa protein (Yew et al.2013 Mol Gen Metab 109:339).

In certain embodiments, provided herein are compositions comprising aplurality of functional erythroid cells comprising a phenylalaninehydroxylase (PAH) receiver, which may be administered to phenylketonuria(PKU) subjects to treat phenylalanine accumulation. The functionalerythroid cells may contain exogenous PAH within their intracellularspace. The intracellularly localized exogenous PAH polypeptide may thenconvert phenylalanine to tyrosine, thereby lowering the levels ofphenylalanine. Phenylalanine crosses the cell membrane, is converted totyrosine, which diffuses back into circulation. This may be sufficientto reduce phenylalanine concentrations in the blood.

3. MNGIE

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for MitochondrialNeurogastrointestinal Encephalopathy (MNGIE). Subjects suffering from orat risk of developing MNGIE may be administered a pharmaceuticalcomposition comprising the synthetic membrane-receiver polypeptidecomplex described herein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising the enzyme thymidine phosphorylase (TP)or a derivative or functional fragment thereof. A suitable receiver maybe exhibited on the surface of the synthetic membrane-receiverpolypeptide complex. A suitable receiver may be contained in theinterior of the synthetic membrane-receiver polypeptide complex. Thesuitable receiver is capable of catalyzing the phosphorylation ofthymidine or deoxyuridine to thymine or uracil.

In MNGIE, aberrant thymidine metabolism leads to impaired replication ormaintenance of mtDNA, causing mtDNA depletion, deletion, or both(Nishino et al. 1999 Science 283:689). The disease is characterized byprogressive gastrointestinal dysmotility and cachexia manifesting asearly satiety, nausea, dysphagia, gastroesophageal reflux, postprandialemesis, episodic abdominal pain and/or distention, and diarrhea;ptosis/ophthalmoplegia or ophthalmoparesis; hearing loss; anddemyelinating peripheral neuropathy manifesting as paresthesias(tingling, numbness, and pain) and symmetric and distal weakness moreprominently affecting the lower extremities. There is no treatment forMNGIE. Management is supportive and includes attention to swallowingdifficulties and airway protection; dromperidone for nausea andvomiting; celiac plexus block with bupivicaine to reduce pain; bolusfeedings, gastrostomy, and parenteral feeding for nutritional support;antibiotics for intestinal bacterial overgrowth; morphine,amitriptyline, gabapentin, and phenytoin for neuropathic symptoms;specialized schooling arrangements; and physical and occupationaltherapy.

4. Lysosomal Enzyme Deficiency

The synthetic complexes described herein can be useful for the treatmentof Lysosomal storage disorders. In one embodiment a syntheticmembrane-receiver polypeptide complex comprises a receiver, e.g., anenzyme that is active in cell lysosomes and can degrade accumulatedtoxic compounds, e.g., proteins, polypeptides, carbohydrates, or lipids,in lysosomes of cells with a deficiency in a lysosomal enzyme. Thereceiver will act by reducing the amount of toxic compound accumulatedin the lysosomes of these cells, thus reducing the burden of thedisease. Lysosomal storage disorders include, but are not limited to,mucopolysaccharidosis I, Gaucher Disease, Fabry Disease, Pompe Diseaseand those listed in table 6 and table 8.

In one embodiment, subjects may be identified as having received orwould benefit from receiving treatment for Gaucher's disease. Subjectssuffering from or at risk of developing Gaucher's disease may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising the enzyme glucocerebrosidase or aderivative or functional fragment thereof. A suitable receiver may beexhibited on the surface of the synthetic membrane-receiver polypeptidecomplex or in the interior of the synthetic membrane-receiverpolypeptide complex. The suitable receiver is capable of cleaving byhydrolysis the beta-glucosidic linkage of the chemical glucocerebroside,a sphingolipid.

Gaucher's disease is caused by a hereditary deficiency of the enzymeglucocerebrosidase. When the enzyme is defective, glucocerebrosideaccumulates in white blood cells, spleen, liver, kidneys, lungs, brain,and bone marrow. The disorder is characterized by bruising, fatigue,anemia, low blood platelets, and enlargement of the liver and spleen.Manifestations may include enlarged spleen and liver, liver malfunction,skeletal disorders and bone lesions that may be painful, severeneurologic complications, swelling of lymph nodes and (occasionally)adjacent joints, distended abdomen, a brownish tint to the skin, anemia,low blood platelets, and yellow fatty deposits on the white of the eye(sclera). Persons affected most seriously may also be more susceptibleto infection.

Several lysosomal storage disorders are addressable by methods oftreatment described herein. For example: In one embodiment the diseaseor condition is aspartylglucosaminuria (208400), the receiver isN-aspartylglucosaminidase or fragment thereof, and the target isglycoproteins. In one embodiment the disease or condition iscerebrotendinous xanthomatosis (cholestanol lipidosis; 213700), thereceiver is sterol 27-hydroxylase or fragment thereof, and the target islipids, cholesterol, and bile acid. In one embodiment the disease orcondition is ceroid lipofuscinosis adult form (CLN4, Kufs' disease;204300), the receiver is palmitoyl-protein thioesterase-1 or fragmentthereof, and the target is lipopigments. In one embodiment the diseaseor condition is ceroid lipofuscinosis infantile form (CLN1,Santavuori-Haltia disease; 256730), the receiver is palmitoyl-proteinthioesterase-1 or fragment thereof, and the target is lipopigments. Inone embodiment the disease or condition is ceroid lipofuscinosisjuvenile form (CLN3, Batten disease, Vogt-Spielmeyer disease; 204200),the receiver is lysosomal transmembrane CLN3 protein or fragmentthereof, and the target is lipopigments. In one embodiment the diseaseor condition is ceroid lipofuscinosis late infantile form (CLN2,Jansky-Bielschowsky disease; 204500), the receiver is lysosomalpepstatin-insensitive peptidase or fragment thereof, and the target islipopigments. In one embodiment the disease or condition is ceroidlipofuscinosis progressive epilepsy with intellectual disability(600143), the receiver is transmembrane CLN8 protein or fragmentthereof, and the target is lipopigments. In one embodiment the diseaseor condition is ceroid lipofuscinosis variant late infantile form (CLN6;601780), the receiver is transmembrane CLN6 protein or fragment thereof,and the target is lipopigments. In one embodiment the disease orcondition is ceroid lipofuscinosis variant late infantile form, Finnishtype (CLN5; 256731), the receiver is lysosomal transmembrane CLN5protein or fragment thereof, and the target is lipopigments. In oneembodiment the disease or condition is cholesteryl ester storage disease(CESD), the receiver is lisosomal acid lipase or fragment thereof, andthe target is lipids and cholesterol. In one embodiment the disease orcondition is congenital disorders of N-glycosylation CDG Ia (solelyneurologic and neurologic-multivisceral forms; 212065), the receiver isphosphomannomutase-2 or fragment thereof, and the target isN-glycosylated protein. In one embodiment the disease or condition iscongenital disorders of N-glycosylation CDG Ib (602579), the receiver ismannose (Man) phosphate (P) isomerase or fragment thereof, and thetarget is N-glycosylated protein. In one embodiment the disease orcondition is congenital disorders of N-glycosylation CDG Ic (603147),the receiver is dolicho-P-Glc:Man9GlcNAc2-PP-dolicholglucosyltransferase or fragment thereof, and the target isN-glycosylated protein. In one embodiment the disease or condition iscongenital disorders of N-glycosylation CDG Id (601110), the receiver isdolicho-P-Man:Man5GlcNAc2-PP-dolichol mannosyltransferase or fragmentthereof, and the target is N-glycosylated protein. In one embodiment thedisease or condition is congenital disorders of N-glycosylation CDG Ie(608799), the receiver is dolichol-P-mannose synthase or fragmentthereof, and the target is N-glycosylated protein. In one embodiment thedisease or condition is congenital disorders of N-glycosylation CDG If(609180), the receiver is protein involved in mannose-P-dolicholutilization or fragment thereof, and the target is N-glycosylatedprotein. In one embodiment the disease or condition is congenitaldisorders of N-glycosylation CDG Ig (607143), the receiver isdolichyl-P-mannose:Man-7-GlcNAc-2-PP-dolichyl-α-6-mannosyltransferase orfragment thereof, and the target is N-glycosylated protein. In oneembodiment the disease or condition is congenital disorders ofN-glycosylation CDG Ih (608104), the receiver isdolichyl-P-glucose:Glc-1-Man-9-GlcNAc-2-PP-dolichyl-α-3-glucosyltransferaseor fragment thereof, and the target is N-glycosylated protein. In oneembodiment the disease or condition is congenital disorders ofN-glycosylation CDG Ii (607906), the receiver isα-1,3-Mannosyltransferase or fragment thereof, and the target isN-glycosylated protein. In one embodiment the disease or condition iscongenital disorders of N-glycosylation CDG IIa (212066), the receiveris mannosyl-α-1,6-glycoprotein-β-1,2-N-acetylglucosminyltransferase orfragment thereof, and the target is N-glycosylated protein. In oneembodiment the disease or condition is congenital disorders ofN-glycosylation CDG IIb (606056), the receiver is glucosidase I orfragment thereof, and the target is N-glycosylated protein. In oneembodiment the disease or condition is congenital disorders ofN-glycosylation CDG IIc (Rambam-Hasharon syndrome; 266265, the receiveris GDP-fucose transporter-1 or fragment thereof, and the target isN-glycosylated protein. In one embodiment the disease or condition iscongenital disorders of N-glycosylation CDG IId (607091), the receiveris β-1,4-galactosyltransferase or fragment thereof, and the target isN-glycosylated protein. In one embodiment the disease or condition iscongenital disorders of N-glycosylation CDG He (608779), the receiver isoligomeric golgi complex-7 or fragment thereof, and the target isN-glycosylated protein. In one embodiment the disease or condition iscongenital disorders of N-glycosylation CDG Ij (608093), the receiver isUDP-GlcNAc:dolichyl-P NAcGlc phosphotransferase or fragment thereof, andthe target is N-glycosylated protein. In one embodiment the disease orcondition is congenital disorders of N-glycosylation CDG Ik (608540),the receiver is β-1,4-mannosyltransferase or fragment thereof, and thetarget is N-glycosylated protein. In one embodiment the disease orcondition is congenital disorders of N-glycosylation CDG Il (608776),the receiver is α-1,2-mannosyltransferase or fragment thereof, and thetarget is N-glycosylated protein. In one embodiment the disease orcondition is congenital disorders of N-glycosylation, type I (pre-Golgiglycosylation defects), the receiver is α-1,2-mannosyltransferase orfragment thereof, and the target is N-glycosylated protein. In oneembodiment the disease or condition is cystinosis, the receiver iscystinosin (lysosomal cystine transporter) or fragment thereof, and thetarget is cysteine. In one embodiment the disease or condition isFabry's disease (301500), the receiver is trihexosylceramideα-galactosidase or fragment thereof, and the target isglobotriaosylceramide. In one embodiment the disease or condition isFarber's disease (lipogranulomatosis; 228000), the receiver isceramidase or fragment thereof, and the target is lipids. In oneembodiment the disease or condition is Fucosidosis (230000), thereceiver is α-L-fucosidase or fragment thereof, and the target is fucoseand complex sugars. In one embodiment the disease or condition isgalactosialidosis (Goldberg's syndrome, combined neuraminidase andβ-galactosidase deficiency; 256540), the receiver is protectiveprotein/cathepsin A (PPCA) or fragment thereof, and the target is lipidsand glycoproteins. In one embodiment the disease or condition isGaucher's disease, the receiver is glucosylceramide β-glucosidase orfragment thereof, and the target is sphingolipids. In one embodiment thedisease or condition is glutamyl ribose-5-phosphate storage disease(305920), the receiver is ADP-ribose protein hydrolase or fragmentthereof, and the target is glutamyl ribose 5-phosphate. In oneembodiment the disease or condition is glycogen storage disease type 2(Pompe's disease), the receiver is alpha glucosidase or fragmentthereof, and the target is glycogen. In one embodiment the disease orcondition is GM1 gangliosidosis, generalized, the receiver isganglioside β-galactosidase or fragment thereof, and the target isacidic lipid material, gangliosides. In one embodiment the disease orcondition is GM2 activator protein deficiency (Tay-Sachs disease ABvariant, GM2A; 272750), the receiver is GM2 activator protein orfragment thereof, and the target is gangliosides. In one embodiment thedisease or condition is GM2 gangliosidosis, the receiver is Gangliosideβ-galactosidase or fragment thereof, and the target is gangliosides. Inone embodiment the disease or condition is infantile sialic acid storagedisorder (269920), the receiver is Na phosphate cotransporter, sialin orfragment thereof, and the target is sialic acid. In one embodiment thedisease or condition is Krabbe's disease (245200), the receiver isgalactosylceramide β-galactosidase or fragment thereof, and the targetis sphingolipids. In one embodiment the disease or condition islysosomal acid lipase deficiency (278000), the receiver is lysosomalacid lipase or fragment thereof, and the target is cholesteryl estersand triglycerides. In one embodiment the disease or condition ismetachromatic leukodystrophy (250100), the receiver is arylsulfatase Aor fragment thereof, and the target is sulfatides. In one embodiment thedisease or condition is mucolipidosis ML II (I-cell disease; 252500),the receiver is N-Acetylglucosaminyl-1-phosphotransfeerase catalyticsubunit or fragment thereof, and the target is N-linked glycoproteins.In one embodiment the disease or condition is mucolipidosis ML III(pseudo-Hurler's polydystrophy), the receiver isN-acetylglucosaminyl-1-phosphotransfeerase or fragment thereof, and thetarget is N-linked glycoproteins. In one embodiment the disease orcondition is mucolipidosis ML III (pseudo-Hurler's polydystrophy) TypeIII-A (252600), the receiver is catalytic subunit or fragment thereof,and the target is N-linked glycoproteins. In one embodiment the diseaseor condition is mucolipidosis ML III (pseudo-Hurler's polydystrophy)Type III-C(252605), the receiver is substrate-recognition subunit orfragment thereof, and the target is N-linked glycoproteins. In oneembodiment the disease or condition is mucopolysaccharidosis MPS I H/S(Hurler-Scheie syndrome; 607015), the receiver is α-1-iduronidase orfragment thereof, and the target is glycosaminoglycans. In oneembodiment the disease or condition is mucopolysaccharidosis MPS I-H(Hurler's syndrome; 607014), the receiver is α-1-iduronidase or fragmentthereof, and the target is glycosaminoglycans. In one embodiment thedisease or condition is mucopolysaccharidosis MPS II (Hunter's syndrome;309900), the receiver is iduronate sulfate sulfatase or fragmentthereof, and the target is glycosaminoglycans. In one embodiment thedisease or condition is mucopolysaccharidosis MPS III (Sanfilippo'ssyndrome) Type III-A (252900), the receiver is Heparan-S-sulfatesulfamidase or fragment thereof, and the target is glycosaminoglycans.In one embodiment the disease or condition is mucopolysaccharidosis MPSIII (Sanfilippo's syndrome) Type III-B (252920), the receiver isN-acetyl-D-glucosaminidase or fragment thereof, and the target isglycosaminoglycans. In one embodiment the disease or condition ismucopolysaccharidosis MPS III (Sanfilippo's syndrome) TypeIII-C(252930), the receiver is acetyl-CoA-glucosaminideN-acetyltransferase or fragment thereof, and the target isglycosaminoglycans. In one embodiment the disease or condition ismucopolysaccharidosis MPS III (Sanfilippo's syndrome) Type III-D(252940), the receiver is N-acetyl-glucosaminine-6-sulfate sulfatase orfragment thereof, and the target is glycosaminoglycans. In oneembodiment the disease or condition is mucopolysaccharidosis MPSI-S(Scheie's syndrome; 607016), the receiver is α-1-iduronidase orfragment thereof, and the target is glycosaminoglycans. In oneembodiment the disease or condition is mucopolysaccharidosis MPS IV(Morquio's syndrome) Type IV-A (253000), the receiver isgalactosamine-6-sulfate sulfatase or fragment thereof, and the target isglycosaminoglycans. In one embodiment the disease or condition ismucopolysaccharidosis MPS IV (Morquio's syndrome) Type IV-B (253010),the receiver is β-galactosidase or fragment thereof, and the target isglycosaminoglycans. In one embodiment the disease or condition ismucopolysaccharidosis MPS IX (hyaluronidase deficiency; 601492), thereceiver is hyaluronidase or fragment thereof, and the target isglycosaminoglycans. In one embodiment the disease or condition ismucopolysaccharidosis MPS VI (Maroteaux-Lamy syndrome; 253200), thereceiver is N-acetyl galactosamine α-4-sulfate sulfatase (arylsulfataseB) or fragment thereof, and the target is glycosaminoglycans. In oneembodiment the disease or condition is mucopolysaccharidosis MPS VII(Sly's syndrome; 253220), the receiver is β-glucuronidase or fragmentthereof, and the target is glycosaminoglycans. In one embodiment thedisease or condition is mucosulfatidosis (multiple sulfatase deficiency;272200), the receiver is sulfatase-modifying factor-1 or fragmentthereof, and the target is sulfatides. In one embodiment the disease orcondition is Niemann-Pick disease type A, the receiver issphingomyelinase or fragment thereof, and the target is sphingomyelin.In one embodiment the disease or condition is Niemann-Pick disease typeB, the receiver is sphingomyelinase or fragment thereof, and the targetis sphingomyelin. In one embodiment the disease or condition isNiemann-Pick disease Type C1/Type D (257220), the receiver is NPC1protein or fragment thereof, and the target is sphingomyelin. In oneembodiment the disease or condition is Niemann-Pick disease Type C2(607625), the receiver is epididymal secretory protein 1 (HE1; NPC2protein) or fragment thereof, and the target is sphingomyelin. In oneembodiment the disease or condition is prosaposin deficiency (176801),the receiver is prosaposin or fragment thereof, and the target issphingolipids. In one embodiment the disease or condition ispycnodysostosis (265800), the receiver is cathepsin K or fragmentthereof, and the target is kinins. In one embodiment the disease orcondition is sandhoffs disease; 268800, the receiver is β-hexosaminidaseB or fragment thereof, and the target is gangliosides. In one embodimentthe disease or condition is saposin B deficiency (sulfatide activatordeficiency), the receiver is saposin B or fragment thereof, and thetarget is sphingolipids. In one embodiment the disease or condition issaposin C deficiency (Gaucher's activator deficiency), the receiver issaposin C or fragment thereof, and the target is sphingolipids. In oneembodiment the disease or condition is Schindler's disease Type I(infantile severe form; 609241), the receiver isN-acetyl-galactosaminidase or fragment thereof, and the target isglycoproteins. In one embodiment the disease or condition is Schindler'sdisease Type II (Kanzaki disease, adult-onset form; 609242), thereceiver is N-acetyl-galactosaminidase or fragment thereof, and thetarget is glycoproteins. In one embodiment the disease or condition isSchindler's disease Type III (intermediate form; 609241), the receiveris N-acetyl-galactosaminidase or fragment thereof, and the target isglycoproteins. In one embodiment the disease or condition is sialidosis(256550), the receiver is neuraminidase 1 (sialidase) or fragmentthereof, and the target is mucopolysaccharides and mucolipids. In oneembodiment the disease or condition is sialuria Finnish type (Salladisease; 604369), the receiver is Na phosphate cotransporter, sialin orfragment thereof, and the target is sialic acid. In one embodiment thedisease or condition is sialuria French type (269921), the receiver isUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, sialinor fragment thereof, and the target is sialic acid. In one embodimentthe disease or condition is sphingolipidosis Type I (230500), thereceiver is ganglioside β-galactosidase or fragment thereof, and thetarget is sphingolipids. In one embodiment the disease or condition issphingolipidosis Type II (juvenile type; 230600), the receiver isganglioside β-galactosidase or fragment thereof, and the target issphingolipids. In one embodiment the disease or condition issphingolipidosis Type III (adult type; 230650), the receiver isganglioside β-galactosidase or fragment thereof, and the target issphingolipids. In one embodiment the disease or condition is Tay-Sachsdisease; 272800, the receiver is β-hexosaminidase A or fragment thereof,and the target is gangliosides. In one embodiment the disease orcondition is Winchester syndrome (277950), the receiver ismetalloproteinase-2 or fragment thereof, and the target ismucopolysaccharides. In one embodiment the disease or condition isWolman's disease, the receiver is lysosomal acid lipase or fragmentthereof, and the target is lipids and cholesterol. In one embodiment thedisease or condition is α-mannosidosis (248500), type I (severe) or II(mild), the receiver is α-D-mannosidase or fragment thereof, and thetarget is carbohydrates and glycoproteins. In one embodiment the diseaseor condition is β-mannosidosis (248510), the receiver is β-D-mannosidaseor fragment thereof, and the target is carbohydrates and glycoproteins.

Selective Starvation of Metabolites

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent cancers.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target metabolite, such as an amino acidcell in a subject (e.g., a human) suffering from or at risk ofdeveloping a cancer. The target metabolite is essential for survival ofthe cancer cell but not for survival of a healthy, normal cell. Incertain embodiments, the cancer cell is thereby selectively starved ofthe critical metabolite but healthy normal cells are spared because themetabolite is non-critical for those cells. The methods includeadministering a pharmaceutical composition comprising a syntheticmembrane-receiver polypeptide complex described herein. Thepharmaceutical composition is administered in an amount effective tosubstantially reduce the circulatory concentration of the targetmetabolite. In certain embodiments, the administration is carried outintravenously. Diseases that benefit from a selective starvation of atarget metabolites include cancers such as acute lymphoblastic leukemia,acute myeloblastic leukemia, pancreatic adenocarcinoma, p53-null solidtumors and those listed in table 6 and table 8.

In specific embodiments, provided are methods of treating cancer thatinclude administering to a subject in need thereof a pharmaceuticalcomposition of erythrocyte cells that comprise a receiver providedherein in an amount sufficient to treat cancer. The compositionscomprising functional erythroid cells that comprise a chemotherapeuticor a receiver polypeptides capable of treating tumors and liquidcancers, may be administered to subjects that exhibit a cancers,including adrenal, anal, bile duct, bladder, bone, central nervoussystem, breast, leukemia, liver, lung, lymphoma, multiple myeloma,osteosarcoma, pancreatic, and those listed in, but not limited to, table6 and table 8.

In one embodiment the disease or condition is acute lymphoblasticleukemia, the receiver is asparaginase or fragment thereof, and thetarget is asparagine.

In one embodiment the disease or condition is acute myeloblasticleukemia, the receiver is asparaginase or fragment thereof, and thetarget is asparagine.

In one embodiment the disease or condition is p53-null solid tumor, thereceiver is serine dehyrdatase or serine hydroxymethyl transferase orfragment thereof, and the target is serine.

In one embodiment the disease or condition is pancreatic adenocarcinoma,the receiver is asparaginase or fragment thereof, and the target isasparagine.

Acute Lymphoblastic Leukemia (ALL)

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for acute lymphoblastic leukemia(ALL). Subjects suffering from or at risk of developing ALL may beadministered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising asparaginase or a derivative orfunctional fragment thereof. A suitable receiver may be exhibited on thesurface or on the unexposed side of the synthetic membrane-receiverpolypeptide complex and may be administered to reduce the concentrationof asparagine in circulation thereby depriving a cancer cell lacking theability to synthesize L-asparagine and relying on the local environmentfor the amino acid of asparagine.

In specific embodiments, compositions comprising a plurality offunctional erythroid cells comprising an asparaginase receiver areprovided. Such compositions may be used to treat subjects that exhibitor are diagnosed with acute lymphoblastic leukemia (ALL).

Tumor cells lack the ability to synthesize L-asparagine and rely ontheir local environment for the amino acid. Asparaginase is an enzymethat can be isolated from both Escherichia coli and Erwiniachrysanthemi. The foreign-sourced asparaginase is subject to immunogenicreactions that can generate life-threatening human anti-bacterialantibody responses (Avramis, Anticancer Res., 2009 January;29(1):299-302). It has provided therapeutic benefit as a stand-aloneenzyme replacement therapy, but inhibitor development is a common resultof chronic treatment.

In specific embodiments, provided herein are compositions comprising aplurality of functional erythroid cells comprising an asparaginasereceiver which may be administered to ALL subjects to deprive the cancercells of asparagine. The functional erythroid cells may containexogenous asparaginase within their intracellular space. Theintracellularly localized exogenous asparaginase polypeptide may thenconvert asparagine to aspartate, thereby lowering the levels ofasparagine. Asparagine crosses the cell membrane, is converted toaspartate, and diffuses back into circulation. This may be sufficient tocreate a local deficiency in the critical nutrient and starving tumorcells.

Diseases and Conditions Associated with Vascular Deficiencies

In some embodiments, the synthetic membrane-receiver polypeptidecomplexes described herein may be used to treat or prevent diseases andconditions associated with vascular deficiencies, e.g., of a vascularprotein. A schematic example of this aspect of the invention is shown inFIG. 13B.

In some embodiments, the surface exposed receiver polypeptide mayinteract with a target substrate and can modify, convert, change orotherwise alter the target substrate. Alternatively, the surface exposedreceiver polypeptide is cleaved from the surface of the syntheticmembrane-receiver complex in response to a specific microenvironment ormolecule. In one embodiment, the receiver's catalytic activity may beinitiated after cleavage.

In some embodiments, the synthetic membrane-receiver complexes comprisea receiver and optionally comprise a payload, such as a therapeuticagent, that can be released upon lysis of the syntheticmembrane-receiver complex. The payload may be an enzyme, protein,antibody, or small molecule. The lytic event may be triggered by astimulus in the microenvironment in which the syntheticmembrane-receiver complex is present. The stimulus may, for example,recruit membrane-targeting enzymes, trigger the complement system tolyse the synthetic membrane-receiver complex, or mark the complex fordestruction. Alternatively, in embodiments in which the syntheticmembrane-receiver complex is generated from a cell, e.g., an erythroidcell, the synthetic membrane-receiver complex may be modified to undergoapotosis when exposed to a specific stimulus or once a certain period oftime has passed. A schematic example is shown in FIG. 13C.

In specific embodiments, methods are provided for reducing thecirculatory concentration of a target vascular protein in a subject(e.g., a human) suffering from or at risk of developing a disease orcondition associated with a vascular deficiency. The methods includeadministering a pharmaceutical composition comprising a syntheticmembrane-receiver polypeptide complex described herein. The syntheticmembrane-receiver polypeptide complex may comprise a receiver that candegrade, cleave, or convert a vascular protein. In some embodiments, afunction of a missing vascular enzyme (a non-target) is restored. Insome embodiments, the amount of the target vascular protein is reducedto effectively restore the homeostatic balance of vascular proteins tolevels effective to treat or prevent the disease or condition. Incertain embodiments, the administration is carried out intravenously.Diseases or conditions associated with vascular deficiencies include,but are not limited to thrombotic thrombocytopenic purpura, hemophiliaA, hemophilia B, von Willebrand disease and those listed in table 6 andtable 8.

In specific embodiments, provided are methods of treating a clottingdisease or anti-clotting disease. The methods include administering to asubject in need thereof a pharmaceutical composition of erythrocytecells that comprise a receiver provided herein in an amount sufficientto treat the clotting disease or anti-clotting disease. The compositionsmay be administered to subjects that exhibit hemophilia type A,hemophilia type B, hemophilia Type C, von Willebrand disease, Factor IIdeficiency, Factor V deficiency, Factor VII deficiency, Factor Xdeficiency, Factor XII deficiency, thrombophilia, pulmonary embolism,stroke, and those disease or deficiencies included in, but not limitedto, table 6 and table 8.

In one embodiment the disease or condition is hemophilia A, the receiveris factor VIII or fragment thereof, and the target is thrombin (factorII a) or factor X.

In one embodiment the disease or condition is hemophilia B, the receiveris factor IX or fragment thereof, and the target is factor XIa or factorX.

In one embodiment the disease or condition is thromboticthrombocytopenic purpura, the receiver is ADAMTS13 or fragment thereof,and the target is ultra-large von Willebrand factor (ULVWF).

Hemophilia

In some embodiments, subjects may be identified as having received orwould benefit from receiving treatment for hemophilia. Subjectssuffering from or at risk of developing hemophilia may be administered apharmaceutical composition comprising the synthetic membrane-receiverpolypeptide complex described herein to treat or prevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising coagulation factor VIII or a derivativeor functional fragment thereof. A suitable receiver may be exhibited onthe surface of the synthetic membrane-receiver polypeptide complex andmay be administered to provide factor VIII function to subjectsexhibiting hemophilia A.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising coagulation factor IX or a derivative orfunctional fragment thereof. A suitable receiver may be exhibited on thesurface of the synthetic membrane-receiver polypeptide complex and maybe administered to provide factor IX function to subjects exhibitinghemophilia B.

Hemophilia is a common bleeding disorder (occurring in approximately1:10,000 males) in which causes severe internal bleeding that oftenleads to death because the patient's blood doesn't clot normally.Hemophilia usually is inherited with patients displaying severeuncontrollable bleeding events beginning at birth and re-occurringthroughout the individual's life. Although there are several types ofclotting factors that work together with platelets to help the bloodcoagulate, people with hemophilia usually have quantitative orqualitative defects in the proteins that encode coagulation factor VIII(hemophilia A) or factor IX (hemophilia B) that prevent normalhemostasis. Hemophilia usually occurs in males because Factors VIII andIX are located on the X chromosome (although with rare exceptionsfemales who inherit a defective X chromosome each from an affectedfather and mother who is a carrier for the disease). About 1 in 10,000individuals are born with hemophilia each year all over the world.

Hemostasis is the complex physiological process that leads to thecessation of bleeding. Platelets, plasma proteins, blood vessels andendothelial cells each play an important role in the events thatimmediately follow tissue injury and which, under normal circumstances,results in the rapid formation of a clot. Central to this is thecoagulation cascade, a series of proteolytic events in which certainplasma proteins (or coagulation factors) are sequentially activated in a“cascade” by another previously activated coagulation factor, leading tothe rapid generation of thrombin. The large quantities of thrombinproduced in this cascade then function to cleave fibrinogen into thefibrin peptides that are required for clot formation.

The coagulation factors circulate as inactive single-chain zymogens, andare activated by cleavage at one or more positions to generate atwo-chain activated form of the protein. Factor VII (FVII), a vitaminK-dependent plasma protein, initially circulates in the blood as azymogen. The FVII zymogen is activated by proteolytic cleavage at asingle site, Arg152-Ile153, resulting is a two-chain protease linked bya single disulphide bond (FVIIa). FVIIa binds its cofactor, tissuefactor (TF), to form a complex in which FVIIa can efficiently activatefactor X (FX) to FXa, thereby initiating the series of events thatresult in fibrin formation and hemostasis.

The blood coagulation pathway, in part, involves the formation of anenzymatic complex of Factor Villa (FVIIIa) and Factor IXa (FIXa) (Xasecomplex) on the surface of platelets. FIXa is a serine protease withrelatively weak catalytic activity without its cofactor FVIIIa. The Xasecomplex cleaves Factor X (FX) into Factor Xa (FXa), which in turninteracts with Factor Va (FVa) to cleave prothrombin and generatethrombin.

About 9 out of 10 people who have hemophilia have type A. Hemophilia Ais a bleeding disorder caused by mutations and/or deletions in theFactor VIII (FVIII) gene resulting in a deficiency of FVIII activity. Insome cases, patients have reduced levels of FVIII due to the presence ofFVIII inhibitors, such as anti-FVIII antibodies. Hemophilia A ischaracterized by spontaneous hemorrhage and excessive bleeding aftertrauma. Over time, the repeated bleeding into muscles and joints, whichoften begins in early childhood, results in hemophilic arthropathy andirreversible joint damage. This damage is progressive and can lead toseverely limited mobility of joints, muscle atrophy and chronic pain(Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96 (2003)).

The disease can be treated by replacement therapy targeting restorationof FVIII activity to 1 to 5% of normal levels to prevent spontaneousbleeding (see, e.g., Mannucci, P. M., et al., N. Engl. J. Med. 344:1773-9 (2001), herein incorporated by reference in its entirety). Thereare plasma-derived and recombinant FVIII products available to treatbleeding episodes on-demand or to prevent bleeding episodes fromoccurring by treating prophylactically. Based on the half-life of theseproducts (10-12 hr) (White G. C., et al., Thromb. Haemost. 77:660-7(1997); Morfmi, M., Haemophilia 9 (suppl 1):94-99; discussion 100(2003)), treatment regimens require frequent intravenous administration,commonly two to three times weekly for prophylaxis and one to threetimes daily for on-demand treatment (Manco-Johnson, M. J., et al, N.Engl. J. Med. 357:535-544 (2007)), each of which is incorporated hereinby reference in its entirety. Such frequent administration is painfuland inconvenient.

Although on-demand treatment is frequently used, there is a trend towardprophylaxis and the prevention of joint damage (Blanchette P, et al.,Haemophilia 2004: 10; 679-683, Manco-Johnson, M J, et al., N. Engl. J.Med. 2007; 357:535-544). Current FVIII products are administered everytwo to three days for prophylaxis due to the relatively short half-lifeof 10-12 hr in order to maintain a FVIII:C above 1% in patients(Morfini, M, Haemophilia 2003; 9 (suppl 1):94-99; discussion 100, WhiteG C, et al, Thromb. Haemost. 1997:77:660-7, Blanchette, P, et al, J.Thromb. Haemost. 2008 August; 6(8): 1319-26). Longer-acting FVIIItherapies that provide prolonged protection from bleeding wouldrepresent an improvement in the quality of life for patients withhemophilia A.

Strategies to extend the half-life of clotting factors includepegylation (Rostin J, et al, Bioconj. Chem. 2000; 11:387-96),glycopegylation (Stennicke H R, et al, Thromb. Haemost. 2008;100:920-8), formulation with pegylated liposomes (Spira J, et al, Blood2006; 108:3668-3673, Pan J, et al, Blood 2009; 114:2802-2811) andconjugation with albumin (Schulte S., Thromb. Res. 2008; 122 Suppl4:S14-9).

Under normal conditions, activated platelets provide the lipid surfacesupporting coagulation. Since platelets are activated by thrombin, whichis formed at sites of vascular injury, coagulation processes arerestricted to the sites of injuries. However, it is undesirable toprovide the body with peptides that are general substitutes forprocoagulant lipids as this would cause systemic coagulation andultimately lead to disseminated intravascular coagulation (DIC).

U.S. Pat. Nos. 7,109,170 and 6,624,289 disclose regions of the FIXaprotease domain that interact with FVIIIa and that comprise the FVIIIabinding site of FIXa. The peptides inhibit binding of FIXa to FVIIIa.The disclosed peptides may be useful as anticoagulants for preventing ortreating thrombosis.

US20010014456A1 discloses binding molecules for human FVIII andFVIII-like proteins. These polypeptides bind FVIII and/or FVIII-likepolypeptides and are useful for the detection and purification of humanFVIII and/or FVIII-like polypeptides from solutions such as blood orconditioned media.

In U.S. Pat. No. 7,033,590 FIX/FIXa activating antibodies and antibodyderivatives are used for increasing the amidolytic activity of FIXa, andfor treating blood coagulation disorders such as hemophilia A andhemorrhagic diathesis.

U.S. Pat. No. 7,084,109 discloses FVIIa antagonists that are peptidesand inhibit FVIIa activity. The peptides may be useful for theprevention of arterial thrombosis in combination with thrombolytictherapy.

Hemophilia can be mild, moderate, or severe, depending on how muchnormal functional clotting factor is present in the blood. About 7 outof 10 people who have hemophilia A have the severe form of the disorder.

Hemophilia B (also known as Christmas disease) is one of the most commoninherited bleeding disorders in the world. It results in decreased invivo and in vitro blood clotting activity and requires extensive medicalmonitoring throughout the life of the affected individual.

In the absence of intervention, the afflicted individual may suffer fromspontaneous bleeding in the joints, which produces severe pain anddebilitating immobility. Bleeding into muscles results in theaccumulation of blood in those tissues. Spontaneous bleeding in thethroat and neck may cause asphyxiation if not immediately treated.Bleeding into the urine, and severe bleeding following surgery, minoraccidental injuries, or dental extractions also are prevalent.

Hemophilia B is caused by a deficiency in Factor IX that may result fromeither the decreased synthesis of the Factor IX protein or a defectivemolecule with reduced activity.

Human FIX, one member of the group of vitamin K-dependent polypeptides,is a single-chain glycoprotein with a molecular weight of 57 kDa, whichis secreted by liver cells into the blood stream as an inactive zymogenof 415 amino acids. It contains 12 γ-carboxy-glutamic acid residueslocalized in the N-terminal Gla-domain of the polypeptide. The Glaresidues require vitamin K for their biosynthesis. Following the Gladomain there are two epidermal growth factor domains, an activationpeptide, and a trypsin-type serine protease domain. Furtherposttranslational modifications of FIX encompass hydroxylation (Asp 64),N-(Asn157 and Asn167) as well as O-type glycosylation (Ser53, Ser61,Thrl59, Thrl69, and Thrl72), sulfation (Tyrl55), and phosphorylation(Serl58). FIX is converted to its active form, Factor IXa, byproteolysis of the activation peptide at Argl45-Alal46 and Argl80-Vall81leading to the formation of two polypeptide chains, an N-terminal lightchain (18 kDa) and a C-terminal heavy chain (28 kDa), which are heldtogether by one disulfide bridge. Activation cleavage of Factor IX canbe achieved in vitro e.g., by Factor XIa or Factor VIIa/TF. Factor IX ispresent in human plasma in a concentration of 5-10 μg/ml. Terminalplasma half-life of Factor IX in humans was found to be about 15 to 18hours (White G C et al. 1997. Recombinant factor IX. Thromb Haemost. 78:261-265; Ewenstein B M et al. 2002. Pharmacokinetic analysis ofplasma-derived and recombinant F IX concentrates in previously treatedpatients with moderate or severe hemophilia B. Transfusion 42: 190-197).

The treatment of hemophilia B occurs by replacement of the missingclotting factor by exogenous factor concentrates highly enriched inFactor IX. However, generating such a concentrate from blood isdifficult. Purification of Factor IX from plasma (plasma derived FactorIX; pdFIX) almost exclusively yields active Factor IX. However, suchpurification of FIX from plasma is very difficult because FIX is onlypresent in low concentration in plasma (Andersson, Thrombosis Research7: 451 459 (1975). Further, purification from blood requires the removalor inactivation of infectious agents such as HIV and HCV. In addition,pdFIX has a short half-life and therefore requires frequent dosing.Recombinant FIX (rFIX) is also available, but suffers from the sameshort half-life and need for frequent dosing (e.g., 2-3 times per weekfor prophylaxis) as pdFIX.

A recombinant FVIIa product is marketed by Novo Nordisk (NovoSeven).Recombinant FVIIa has been approved for the treatment of hemophilia A orB patients that have inhibitors to FVIII or FIX, and is used to stopbleeding episodes or prevent bleeding associated with trauma and/orsurgery, as well as being approved for the treatment of patients withcongenital FVII deficiency. FVIIa therapy leaves significant unmetmedical need, because an average of 3 doses of FVIIa over a 6 hour timeperiod are required to manage acute bleeding episodes in hemophiliapatients.

Complications of replacement therapy include developing antibodiesresponse to the normal therapeutic protein that is foreign to thepatient's immune system (known as inhibitor formation), which ultimatelyleads to inactivation or destruction of the clotting factor anduncontrolled bleeding in about 30% of patients, developing viralinfections from human clotting factors (from blood contaminated with HIVor Hepatitis from infected blood donors especially in third worldcountries), very expensive costs of the replacement protein which has avery short half-life (days) which requires frequent re-administration tosubside a severe vascular injury and damage to joints, muscles, or otherparts of the body resulting from delays in treatment.

In specific embodiments, provided herein are platelets comprising areceiver polypeptide capable of treating or preventing clottingdiseases, including hemophilia. Suitable receiver polypeptides includeclotting factors, e.g., Factor VIII and/or Factor IX. Human Factor VIIIhas the accession number NM 000132.3 and Human Factor IX has theaccession number NM 000133.3.

In some embodiments, methods of treatment of hemophilia are providedcomprising co-administration of one or more recombinant factors (e.g.,recombinant FIX, FIXa, FVIII, and FVIIa) and the syntheticmembrane-receiver complex described herein, wherein co-administrationincludes administration of the recombinant factor before, after orconcurrent with administration of the synthetic membrane-receivercomplex.

In some embodiments, methods of treatment of viral infectious diseasesare provided comprising administration of a pharmaceutical compositioncomprising one or more recombinant factors (e.g., recombinant FIX, FIXa,FVIII, and FVIIa) and the synthetic membrane-receiver complex describedherein.

In some embodiments, a single treatment is utilized to provide long-termprotection against episodes of bleeding. In some embodiments that treathemophilia, treatment is performed on a regular basis (e.g., weekly,monthly, yearly, once every 2, 3, 4, 5 or more years, and the like) inorder to prevent episodes of bleeding. In some embodiments, treatment isonly administered when episodes of abnormal bleeding occur (e.g.,following accidents, prior to or following surgery, etc,). In someembodiments, maintenance therapy is administered in combination withextra therapy when episodes of abnormal bleeding occur.

Thrombotic Thrombocytopenic Purpura

In some embodiment, subjects may be identified as having received orwould benefit from receiving treatment for Thrombotic ThrombocytopenicPurpura (TTP). Subjects suffering from or at risk of developing TTP maybe administered a pharmaceutical composition comprising the syntheticmembrane-receiver polypeptide complex described herein to treat orprevent disease.

In one embodiment, the synthetic membrane-receiver polypeptide complexcomprises a receiver comprising the protease ADAMTS13 or a derivative orfunctional fragment thereof. A suitable receiver may be exhibited on thesurface of the synthetic membrane-receiver polypeptide complex. Thesuitable receiver is capable of cleaving ultra-large von WillebrandFactor (UL-VWF) multimers into smaller multimers.

Circulating multimers of UL-VWF increase platelet adhesion to areas ofendothelial injury, particularly at arteriole-capillary junctions. Redblood cells passing the microscopic clots are subjected to shear stresswhich damages their membranes, leading to intravascular hemolysis, whichin turn leads to anaemia and schistocyte formation. Reduced blood flowdue to thrombosis and cellular injury results in end organ damage.Current therapy is based on support and plasmapheresis to replenishblood levels of the enzyme.

EXAMPLES Example 1: Gene Assembly

DNA encoding the following genes—glycophorin A (Uniprot ID P02724), Kell(Uniprot ID P23276), antibody scFv against hepatitis B surface antigen(Bose et al. 2003 Mol Immunol 40(9):617, GenBank ID AJ549501.1),adenosine deaminase (Uniprot ID P00813), phenylalanine hydroxylase fromChromobacterium violaceum (GenBank ID AF146711.1), complement receptor 1(Uniprot ID P17927), CD46 (GenBank: BAA12224.1), CD55 (Uniprot IDP08174), CD59 (Uniprot ID P13987), green fluorescent protein (Uniprot IDP42212), thymidine phosphorylase (Uniprot ID P19971), glucocerebrosidase(Uniprot ID P04062), beta2 glycoprotein 1 (Uniprot ID P02749),phospholipase a2 receptor (Uniprot ID Q13018), collagen alpha-3(IV)(Uniprot ID Q01955), serum amyloid P (Uniprot ID P02743), lipoproteinlipase (Uniprot ID P06858), asparaginase (Uniprot ID P00805), factor IX(Uniprot ID F2RM35), ADAMTS13 (Uniprot ID Q76LX8)—were purchased as cDNAfrom Dharmacon (GE Life Sciences) or synthesized de novo by DNA2.0 andGenscript.

1. Single Gene Cloning (CR1)

Genes were assembled into expression vectors by standard molecularbiology methods known in the art. The gene for complement receptor 1(CR1) was synthesized by a commercial vendor (DNA2.0) and supplied in astandard cloning vector (pJ series). The gene was amplified out of thepJ vector by polymerase chain reaction (PCR) using oligos withnon-homologous terminal sequences to prepare for insertion into themammalian expression vector (System Biosciences, pM series): theupstream oligo consisted of 25 nt homologous to the upstream pMinsertion site and 25 nt homologous to the start of CR1; the downstreamoligo consisted of 25 nt homologous to the downstream pM insertion siteand 25 nt homologous to the end of CR1. The amplified product waspurified by gel electrophoresis (Qiagen). The pM vector was linearizedby PCR with tail-to-tail oligos homologous to the upstream anddownstream insertion sites and purified by PCR purification (Qiagen).The CR1 amplicon was ligated into the linearized pM vector by Gibsonassembly, described in detail in Gibson 2011, Methods Enzymology Vol498, p. 394. Sequences were confirmed by Sanger sequencing.

2. Fusion of Two Genes (membrane Kell-scFv)

The gene for Kell was purchased as cDNA and supplied in a standardcloning vector (pJ series). The gene for an antibody scFv specific tohepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617) was synthesized by a commercial vendor (DNA2.0) andsupplied in a standard cloning vector (pJ series). The genes wasamplified out of the pJ vectors by polymerase chain reaction (PCR) usingoligos with non-homologous terminal sequences to prepare for insertioninto the mammalian expression vector (System Biosciences, pM series).Kell was amplified with an upstream oligo consisting of 25 nt homologousto the upstream pM insertion site and 25 nt homologous to the 5′terminus of Kell, and a downstream oligo consisting of 25 nt homologousto the 5′ terminus of scFv and 25 nt homologous to the 3′ terminus ofKell. scFv was amplified with an upstream oligo consisting of 25 nthomologous to the 3′ terminus of Kell insertion site and 25 nthomologous to the 5′ terminus of scFv, and a downstream oligo consistingof 25 nt homologous to the downstream pM insertion site and 25 nthomologous to the 3′ terminus of scFv. The amplified products werepurified by gel electrophoresis (Qiagen). The pM vector was linearizedby PCR with tail-to-tail oligos homologous to the upstream anddownstream insertion sites and purified by PCR purification (Qiagen).The Kell and scFv amplicons were ligated into the linearized pM vectorby one-pot Gibson assembly, described in detail in Gibson 2011, MethodsEnzymology Vol 498, p. 394. Sequences were confirmed by Sangersequencing.

3. Linker-Assembly Between Genes (Kell-Scfv)

The gene for Kell was purchased as cDNA and supplied in a standardcloning vector (pJ series). The gene for an antibody scFv specific tohepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617) was synthesized by a commercial vendor (DNA2.0) andsupplied in a standard cloning vector (pJ series). The genes wasamplified out of the pJ vectors by polymerase chain reaction (PCR) usingoligos with non-homologous terminal sequences to prepare for insertioninto the mammalian expression vector (System Biosciences, pM series).Kell was amplified with an upstream oligo consisting of 25 nt homologousto the upstream pM insertion site and 25 nt homologous to the 5′terminus of Kell; and a downstream oligo consisting of 25 nt homologousto the 5′ terminus of scFv, 24 nt encoding a (GlyGlyGlySer)×2 (SEQ IDNO: 23) spacer, and 25 nt homologous to the 3′ terminus of Kell. scFvwas amplified with an upstream oligo consisting of 25 nt homologous tothe 3′ terminus of Kell insertion site, 24 nt encoding a(GlyGlyGlySer)×2_(SEQ ID NO: 23) spacer, and 25 nt homologous to the 5′terminus of scFv; and a downstream oligo consisting of 25 nt homologousto the downstream pM insertion site and 25 nt homologous to the 3′terminus of scFv. The amplified products were purified by gelelectrophoresis (Qiagen). The pM vector was linearized by PCR withtail-to-tail oligos homologous to the upstream and downstream insertionsites and purified by PCR purification (Qiagen). The Kell and scFvamplicons were ligated into the linearized pM vector by one-pot Gibsonassembly, described in detail in Gibson 2011, Methods Enzymology Vol498, p. 394. Sequences were confirmed by Sanger sequencing.

4. Epitope Tag Attachment (Kell-scFv)

The gene for Kell was purchased as cDNA and supplied in a standardcloning vector (pJ series). The gene for an antibody scFv specific tohepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617) was synthesized by a commercial vendor (DNA2.0) andsupplied in a standard cloning vector (pJ series). The genes wasamplified out of the pJ vectors by polymerase chain reaction (PCR) usingoligos with non-homologous terminal sequences to prepare for insertioninto the mammalian expression vector (System Biosciences, pM series).Kell was amplified with an upstream oligo consisting of 25 nt homologousto the upstream pM insertion site and 25 nt homologous to the 5′terminus of Kell; and a downstream oligo consisting of 25 nt homologousto the 5′ terminus of scFv, 24 nt encoding a (GlyGlyGlySer)×2 (SEQ IDNO: 23) spacer, and 25 nt homologous to the 3′ terminus of Kell. scFvwas amplified with an upstream oligo consisting of 25 nt homologous tothe 3′ terminus of Kell insertion site, 24 nt encoding a(GlyGlyGlySer)×2 (SEQ ID NO: 23) spacer, and 25 nt homologous to the 5′terminus of scFv; and a downstream oligo consisting of 25 nt homologousto the downstream pM insertion site, the 27 nt sequencetacccctatgacgtgcccgactatgcc (Seq. ID No. 8) encoding an HA epitope tag,and 25 nt homologous to the 3′ terminus of scFv. The amplified productswere purified by gel electrophoresis (Qiagen). The pM vector waslinearized by PCR with tail-to-tail oligos homologous to the upstreamand downstream insertion sites. The downstream primer additionallycontained the 27 nt sequence tacccctatgacgtgcccgactatgcc (Seq. ID No. 8)encoding an HA epitope tag. The linearized vector was purified by PCRpurification (Qiagen). The Kell and scFv amplicons were ligated into thelinearized pM vector by one-pot Gibson assembly, described in detail inGibson 2011, Methods Enzymology Vol 498, p. 394. Sequences wereconfirmed by Sanger sequencing.

5. Fusion of Two Genes (Reporter Assembly) (GPA-HA)

The genes for complement receptor 1 (CR1) and green fluorescent protein(GFP) were synthesized by a commercial vendor (DNA2.0) and supplied instandard cloning vectors (pJ series). The CR1 gene was amplified out ofthe pJ vector by polymerase chain reaction (PCR) using oligos withnon-homologous terminal sequences to prepare for insertion into themammalian expression vector (System Biosciences, pM series): theupstream oligo consisted of 25 nt homologous to the upstream pMinsertion site and 25 nt homologous to the start of CR1; the downstreamoligo consisted of 54 nt homologous to the viral-derived T2A sequencegagggcagaggaagtcttctaacatgcggtgacgtggaggsgsstcccggccct (Seq. ID No. 7).The GFP gene was amplified out of the pJ vector by polymerase chainreaction (PCR) using oligos with non-homologous terminal sequences toprepare for insertion into the mammalian expression vector (SystemBiosciences, pM series): the upstream oligo consisted of 54 nthomologous to the viral-derived T2A sequencegagggcagaggaagtcttctaacatgcggtgacgtggaggsgsstcccggccct (Seq. ID No. 7)and 25 nt homologous to the start of GFP; the downstream oligo consistedof 25 nt homologous to the downstream pM insertion site and 25 nthomologous to the end of GFP. The amplified products were purified bygel electrophoresis (Qiagen). The pM vector was linearized by PCR withtail-to-tail oligos homologous to the upstream and downstream insertionsites and purified by PCR purification (Qiagen). The CR1 and GFPamplicons were ligated together and into the linearized pM vector byGibson assembly, described in detail in Gibson 2011, Methods EnzymologyVol 498, p. 394. Sequences were confirmed by Sanger sequencing.

Example 2: mRNA Assembly

A gene of interest is cloned into the multiple cloning site of the pSP64vector (Promega) using standard molecular biology methods. The vector isdigested with EcoRI (NEB) to generate a linearized dsDNA vectorcontaining the SP6 promoter, gene of interest, and 30 nucleotide longpoly-A tail. mRNA is synthesized by reaction with SP6 RNA polymerase(Promega) according to manufacturer's instructions, includingrecommended concentrations of 5′ cap analog (ARCA) to synthesize cappedmRNA transcript. The reaction mixture is then treated with DNAse todigest the template vector (Riboprobe from Promega) and the mRNA ispurified using the EZNA MicroElute RNA Clean-Up kit (Omega).

Example 3: Cell Culture

1. Human Red Blood Cells (RBCs)

CD34 cells are isolated from peripheral blood by supermagnetic microbeadselection by the use of Mini-MACS columns (Miltenyi Biotec; 94%+/−3%purity). The cells are cultured in erythroid differentiation medium(EDM) on the basis of IMDM supplemented with stabilized glutamine, 330μg/mL holo-human transferrin, 10 μg/mL recombinant human insulin, 2IU/mL heparin, and 5% solvent/detergent virus-inactivated plasma. Theexpansion procedure comprises 3 steps. In the first step (day 0 to day7), 10̂4/mL CD34+ cells are cultured in EDM in the presence of 1 μMhydrocortisone, 100 ng/mL SCF, 5 ng/mL IL-3, and 3 IU/mL EPO. On day 4,1 volume of cell culture is diluted in 4 volumes of fresh mediumcontaining SCF, IL-3, EPO, and hydrocortisone. In the second step (day 7to day 11), the cells are resuspended at 10̂5/mL in EDM supplemented withSCF and EPO. In the third step (day 11 to day 18), the cells arecultured in EDM supplemented with EPO alone. Cell counts are adjusted to7.5×10̂5 to 1×10̂6 and 5-10×10̂6 cells/mL on days 11 and 15, respectively.Beyond day 18, the culture medium containing EPO is renewed twice aweek. The cultures are maintained at 37° C. in 5% CO2 in air.

2. Mouse Red Blood Cells

Methods of culturing mouse erythroid cells from mouse fetal livererythroid progenitors are known in the art, see e.g., Shi et al. 2014,PNAS 2014 111(28):10131.

Mouse erythroid progenitors are isolated from fetal livers. Fetal liversare purchased from Charles River Labs. Livers are put in 1 ml PBS onice. Pipette up and down to get a single-cell suspension solution andpass by a 70 um strainer (BD Falcon 35-2235). Rinse the mesh with 1 mlPBS. Combine the flow through (1 ml per embryo). Pellet the cells at 1.5k RPM for 5 min, re-suspend with red cell lysis buffer (AmmoniumChloride Solution from Stemcell), and incubate on ice for 10 mins.Pellet the cells at 1.5 k RPM for 5 min, remove the lysis buffer, andre-suspend with 10 ml PBS-2% FBS. Add chromPure Rat IgG (JacksonImmunoResearch, #012-000-003) at 50 ul/mouse and incubate at 4 C for 5min Add Biotinylated anti-mouse TER119 (BD Pharmingen, #553672) at (at 1ul/1*10̂6 cells) and incubate at 4 C for 15 min. Add Ms Lineage Panel(Fisher Scientific (Thermo Fisher Scientific) # BDB559971) to the cellsat (2 ul/1*10̂6 cells) and incubate at 4 C for 15 min Washing once with10× volume of PBS/and Spin the cells with 1.5 k RPM for 5 min at 4degree. Add Streptavidin Particles Plus—DM (magnetic beads) (BDPharmigen, #557812) (5 ul/1*10̂6 cells) and incubate at 4 C for 30 min.Prepare 2-4 FACS tubes on a magnetic holder. Aliquot 2 ml cells intoeach tube (4 ml in total), and carefully take the cells out of the tubeand put into the other tube on the other side avoiding the disruption ofthe magnetic stick beads. Repeat the same procedure and take the Ter119negative and linkage negative cells to a new tube. Concentrate thecells, and resuspend the cells with 50-100 ul PBS (2% FBS).

Purified erythroid progenitors are cultured in differentiation mediumcomprising (for 40 mL): IMDM: 29 ml, FBS (Stem Cell): 6 ml (Final 15%),10% BSA in IMDM (Stem Cell): 4 ml (Final 1%), 10 mg/ml Holo-transferrin:2000 ul (Final: 500 ug/ml), 100*L-Glutamine: 400 ul, 100*penicillinstreptomycin: 400 ul, 10 U/ul Epo: 2 ul (Final: 0.5 U/ml), 10 mg/mlInsulin: 40 ul (Final: 10 ug/ml). Culture 2*10̂5 cells/ml in thedifferentiation medium in 24 wells plate at 37 C. After a total cultureof 44-48 hours, analyses are performed, for example by flow cytometry asperformed herein. Enucleated red blood cells are gated out using(Hoechst stain) for differentiation profile analysis. A successfulculture will yield 16 fold increase.

3. Platelets

Donated CD34+ cells are acquired from the Fred Hutchinson CancerResearch Center. The CD34+ enriched cells are plated in a serum-freemedium at 2-4×10̂4 cells/mL and medium refreshment is done on day 4 byadding an equal volume of media. On day 6, cells are counted andanalyzed: 1.5×10̂5 cells are washed and placed in 1 mL of the same mediumsupplemented with a cytokine cocktail consisting of TPO 30 ng/mL, SCF 1ng/mL, interleukin (IL)-6 7.5 ng/mL and IL-9 13.5 ng/mL] to inducemegakaryocyte differentiation. At day 10, ½-¼ of the suspension cultureis replaced with fresh medium. All cytokines are purchased fromPeprotech. The cultures are incubated in a humidified atmosphere (10%CO2) at 39° C. for the first 6 days of culture and 37° C. for the last 8days. Viable nucleated cells are counted with a hemocytometer (0.4%trypan blue; Invitrogen, Burlington, ON, Canada).

Clonogenic progenitor cells (CPC) are assayed using MethoCult H4436 formyeloid CPC, and MegaCult-C for colony-forming unit-megakaryocyte(CFU-Mk), according to manufacturer's instructions (StemCellTechnologies, Vancouver, BC, Canada). To assess differentiation, cellsare stained with antibodies against CD61m CD42b, CD41, CD61, and CD49bby flow cytometry using a FACS-Calibur (Becton Dickinson). For cellcycle analysis, cells are rinsed with phosphate-buffered saline (PBS),fixed with formaldehyde 2% (Sigma, St Louis, Mo., USA) for 5 min andpermeabilized with 0.1% of Triton X-100 (Bio-Rad, Hercules, Calif.,USA). Cells are then marked with mAb-Ki-67-FITC (BD Bioscience, SanJose, Calif., USA), washed and resuspended in 0.5 mL PBS-1% fetal bovineserum (FBS)-0.01% azide 7-amino-actinomycin D (7-AAD) following themanufacturer's instructions (BD Biosciences).

Example 4: Cell Isolation

1. Primary RBCs

Whole blood is collected using aseptic techniques in tubes containinglow molecular weight heparin, dalteparin sodium (9 units/mL blood).Blood is centrifuged at 5000×g for 5 minutes and after removal of plasmaand buffy coat (both can be retained for later use), the erythrocytesare washed twice in cold (4 C) phosphate buffered saline (PBS) withcentrifugation. The resultant red blood cell population is stored at 4 Cin CPDA-1 anticoagulant or a glycerol solution for long-termpreservation.

2. Primary Platelets

Whole blood (40 ml) is collected in 3.8% sodium citrate (1:9 citrate toblood vol/vol) from healthy individuals under an appropriate IRBprotocol. Blood is centrifuged at 200 g for 15 minutes to isolateplatelet-rich plasma (PRP). Platelets are then washed in modifiedTyrode's buffer (containing 138 mM NaCl, 5.5 mM dextrose, 12 mM NaHCO3,0.8 mM CaCl2, 0.4 mM MgCl2, 2.9 mM KCl2, 0.36 mM Na2HPO4 and 20 mMHepes, pH 7.4) in presence of 1 μM prostaglandin 12, and resuspended inthe same buffer.

Example 5: Irradiation of Primary or Cultured Cell

Irradiation of a population of synthetic membrane-receiver complexes canbe performed to ensure that they are incapable of replication. Suchprotocols are similar to those known in the art for irradiating cells,e.g., primary red blood cells. Briefly, one unit (350 ml) of whole bloodis taken and divided into two aliquots of 175 ml each, 10 such units arethus divided into 20 aliquots. One aliquot (175 ml) from each unit ofblood is subjected to gamma irradiation of 25 Gy, and not exceeding 50Gy, by a self-contained gamma cell irradiator (GammaCell 1000,Theratronics). The blood is then stored at 4 C under conventional bloodbanking conditions. Sampling is done from these 10 irradiated and 10non-irradiated blood bags on days 0, 7, 14, and 21 with the help ofsampling site coupler (Fenwal, USA). Tests for cell proliferation areconducted, including a thymidine incorporation assay to quantify anymitotic potential. Supernatant is assayed for free hemoglobin byabsorbance spectroscopy, and for free lactate dehydrogenase bycolorimetric assay (Pierce) to evaluate levels of cell lysis.

Example 6: Enucleation of Erythroid Cells

Erythroid cells are grown to semiconfluence (1 to 4×10̂4 cells per cm2)on 12-mm diameter coverslips coated with collagen in IMDM mediumsupplemented with 100 units/mi of penicillin and 100 units/ml ofstreptomycin. The collagen is necessary to prevent all the cells fromfalling off the coverslip during centrifugation. Cells are grown tomonolayers (5×104 cells per cm2) on coverslips either in the same mediumor in Dulbecco's modified Eagle's medium with 10% calf serum. It is notnecessary to coat the cell coverslips with collagen. In order toenucleate the cells, the coverslips are inverted (cell side down) andplaced into the bottom of 15-ml Corex centrifuge tubes containing 2-5 mlof medium with 10 g of cytochalasin B per ml. The centrifuge tubes withthe coverslips are placed immediately into a Sorvall RC-2 centrifugethat has been warmed to 37 C by spinning the (SS 34) rotor with the headin place for about 1 hr at 10,000 rpm (with the temperature regulatorset at 37-39°). The length of time and speed of centrifugation arecrucial factors for successful enucleation. Cells are spun at 9000 rpmfor 1 hr at 37±20 and cells are spun at 6500 rpm for 50 min at 37±−20.After centrifugation, the coverslips are removed from the centrifuge andplaced cell side up into 35-mm (Falcon) tissue culture dishes(Biolquest) containing 3 ml of medium without cytochalasin B. Within30-60 min at 370, the cells are morphologically normal and 90-99% lackednuclei. Enucleated cells are removed from the coverslips by treatmentwith trypsin-EDTA (Grand Island Biological Co.) and the cells aresuspended in normal medium. The enucleated cells are then replated insmall drops on 22-mm2 coverslips kept in 35-mm tissue culture dishes andplaced in an incubator. At time intervals after replating, thecoverslips are mounted on slides (12) and observations on the enucleatesare made with Zeiss phase contrast, polarized light, and Nomarskioptics.

Example 7: Contacting of Cells

1. Nucleic Acid—Transfection

The nucleic acid of interest is scaled up to provide approximately 5 ugnucleic acid per 10̂5 complexes to be loaded, e.g., a cell, such as anerythroid cell, a platelet, or a hematopoietic precursor cell. Thenucleic acid is diluted in Opti-MEM Medium (Life Technologies) at aratio of 1 ug to 50 uL medium. The diluted nucleic is then combined witha transfection reagent (Trans-IT for DNA, Trans-IT mRNA for mRNA,Trans-IT siRNA for siRNA, Mirus Bio) at a 1:1 volume ratio and allowedto form complexes for 5 minutes at room temperature. The nucleic acidcomplex is added to cells for 12-24 hours. Optionally, after this periodof time, the media can be exchanged with fresh media such that thetransfection reagents are no longer present.

2. Nucleic Acid—Viral Transduction

The gene of interest is cloned into the multiple cloning site oflentivirus vector pCDH with the MSCV promoter sequence from SystemBiosciences.

Lentivirus is produced in 293T cells by transfecting the cells withlipofectamine 5×10̂6 293T cells (Lenti-X 293T Cell Line, Clontech catalog#632180) are plated in a P10 petri dish the day before transfection.Cell confluency should be around 70%. One plate is transfected perconstruct. 20 μl (10 μg) pPACKH1 (System Biosciences) plasmid mix+2 μglenti construct+20 μl Plus reagent (LifeTechnologies, Catalog#11514-015) are combined in 400 μl Optimem and incubated 15 min at RT.30 μl of LF2000 (LifeTechnologies, Catalog #11668-019) is diluted into400 μl Optimem, added dropwise to DNA mix, and incubated for 15 min RT.DNA mix is added to cells (cells are in 9 ml of Optimem). Cells areincubated for 6 hours and then the medium is changed to DMEM/10% FBS.The virus supernatant is collected 48 hours post-transfection bycentrifugation at 1,500 rpm for 5 minutes. The supernatant is collectedand frozen in 1 ml aliquots at −80° C.

Target cells are transduced at day 3-7 of the culture process describedherein. 5×10̂5 cultured cells are plated in 500 μL of medium containing20 μg/mL polybrene in a 24-well plate. For each virus, cells aretransduced in triplicate wells. Virus supernatant is added in another500 μL of medium and the sample is mixed by pipetting. Infection isachieved by spinoculation, spinning the plate at 2000 rpm for 90 minutesat room temperature. After spinoculation, the cells are incubated at 37C overnight, and the next day 1 mL of fresh IMDM medium with appropriatecytokines is added.

3. Nucleic Acid—Cationic Polymer

An mRNA encoding the transgene of interest, and including an upstreampromoter sequence and a downstream poly A tail, can be purchased frommultiple commercial vendors (e.g., IDT-DNA, Coralville Iowa). RNAtransfections are carried out using RNAIMax (Invitrogen, Carlsbad,Calif.) or TRANSIT-mRNA (Mims Bio, Madison, Wis.) cationic lipiddelivery vehicles. RNA and reagent are first diluted in Opti-MEM basalmedia (Invitrogen, Carlsbad, Calif.). 100 ng/uL RNA is diluted 5× and 5μL, of RNAIMax per μg of RNA is diluted 10×. The diluted components arepooled and incubated 15 minutes at room temperature before they aredispensed to culture media. For TRANSIT-mRNA transfections, 100 ng/uLRNA is diluted 10× in Opti-MEM and BOOST reagent is added (at aconcentration of 2 μL, per μg of RNA), TRANSIT-mRNA is added (at aconcentration of 2 μL, per μg of RNA), and then the RNA-lipid complexesare delivered to the culture media after a 2-minute incubation at roomtemperature. RNA transfections are performed in Nutristem xenofree hESmedia (STEMGENT®, Cambridge, Mass.) or Opti-MEM plus 2% FBS. Successfulintroduction of the mRNA transcript into host cells can be monitoredusing various known methods, such as a fluorescent label or reporterprotein, such as Green Fluorescent Protein (GFP). Successfultransfection of a modified mRNA can also be determined by measuring theprotein expression level of the target polypeptide by e.g., WesternBlotting or immunocytochemistry. Similar methods may be followed forlarge volume scale-up to multi-liter (5-10,000 L) culture formatfollowing similar RNA-lipid complex ratios.

4. Nucleic Acid—Electroporation

mRNA encoding the transgene of interest, and including an upstreampromoter sequence and a downstream poly A tail, can be purchased frommultiple commercial vendors (e.g., IDT-DNA, Coralville Iowa).Electroporation parameters are optimized by transfecting erythroidlineage cells with mRNA transcripts and measuring transfectionefficiency by quantitative RT-PCR with primers designed to specificallydetect the exogenous transcripts. For certain cells preparations,discharging a 150 uF capacitor into 2.5×10̂6 cells suspended in 50 μl ofOpti-MEM (Invitrogen, Carlsbad, Calif.) in a standard electroporationcuvette with a 2 mm gap is sufficient for repeated delivery in excess of10,000 copies of modified mRNA transcripts per cell, as determined usingthe standard curve method, while maintaining high viability (>70%). Celldensity may vary from 1×10̂6 cell/50 μl to a density of 2.5×10̂6 cells/500and require from 110V to 145V to transfect cells with similarefficiencies measured in transcript copies per cell. Large multi-liter(5-10,000 L) electroporation may be performed similar to large volumeflow electroporation strategies similar to methods described with theabove described constraints (Li et al., 2002; Geng et al., 2010).

5. Polypeptide—Liposome

Cells, including primary terminally-differentiated cells e.g.,erythrocytes, can be loaded with exogenous protein on their surface andin their cytoplasm. The loading of proteins can be performed usingliposomes.

Lipids (Pro-Ject reagent, Pierce) in organic solvent were dried undernitrogen into a thin film in glass scintillation vial. Approximately 2uL lipids were used per 10̂5 cells. Polyclonal mouse IgG (Abcam) waslabeled with Dylight-650 (Pierce) per manufacturer's instructions.Protein solution at 0.1 mg/mL in PBS was added to the dried lipidmixture. The solution was pipetted several times, incubated for 5minutes at room temperature, then vortexed vigorously to generateencapsulating liposomes. Serum-free medium was added to bring the totalvolume to 500 uL per 10̂5 cells. The liposomal mixture was then incubatedwith the cells for 3-4 hours at 37 C.

FIG. 1A-FIG. 1F shows the loading of an exogenous protein, in this casefluorescently-labeled IgG, into primary erythrocytes with liposomes. Theloading is measured by flow cytometry. The loading is dose-dependent, as0.06% of cells are fluorescent without liposomes, ˜60% of cells arefluorescent at a low liposome dose, and ˜85% of cells are fluorescent ata high liposome dose. The data in FIG. 1A-FIG. 1F is strong proof thatexogenous proteins can be loaded into erythroid cells with liposomes.

6. Polypeptide—Mechanical Disruption

Cells may be loaded using a microfluidic device containing 1 μm, 2 μm, 3μm, 4 μm, 5 μm, 10 μm wide channels that transiently porate the cells,allowing a payload to enter when the cells are pressured through thesystem.

The silicon-based devices are fabricated at the Massachusetts Instituteof Technology microfabrication facility using photolithography and deepreactive ion etching techniques. In this process, 6″ silicon wafers witha 450-μm thickness are treated with hexamethyldisilazane, spin coatedwith photoresist (OCG934; FujiFilm) for 60 s at 3,000 rpm, exposed to UVlight (EV1; EVG) through a chrome mask with the constriction channeldesign, and developed in AZ405 (AZ Electronic Materials) solution for100 s. After 20 min of baking at 90° C., the wafer is etched by deepreactive ion etching (SPTS Technologies) to the desired depth (typically15 μm). The process is repeated on the opposite side of the wafer (i.e.,the one not containing the etched channels) using a different mask,which contains the access hole patterns, and using a thicker photoresistAZ9260 (AZ Electronic Materials). Wet oxidation is then used to grow100-200 nm of silicon oxide before the wafer is anodically bonded to aPyrex wafer and diced into individual devices. Before each experiment,devices are visually inspected and mounted onto a holder with inlet andoutlet reservoirs (all designed in-house and produced by Firstcut).These reservoirs interface with the device using Buna-N O-rings(McMaster-Carr) to provide proper sealing. The inlet reservoir isconnected to a home-made pressure regulator system using Teflon tubingto provide the necessary driving force to push material through thedevice. A population of erythroid cells is first suspended in thedesired delivery buffer [growth medium, PBS, or PBS supplemented with 3%FBS and 1% F-68 Pluronics (Sigma)], mixed with the desired deliverymaterial, and placed in the device's inlet reservoir. This reservoir isconnected to a compressed air line controlled by a regulator, and theselected pressure (0-70 psi) is used to drive the fluid through thedevice. Treated cells are then collected from the outlet reservoir.Cells are incubated at room temperature in the delivery solution for5-20 min after treatment to ensure hole closure before being subjectedto any further treatment. To deliver fluorescently labeled phenylalanineammonia hydroxylase (PAH), the experiments are conducted as describedabove such that the delivery buffer contained 0.1-0.3 mg/mL PAH. GFPknockdown is measured as the percentage reduction in a cell population'saverage fluorescence intensity relative to untreated controls.

7. Polypeptide—Surface Conjugation

The cell surface is treated with Traut's reagent (2-iminothiolane HCl,Pierce) to thiolate primary amines Traut's reagent is dissolved in Trisbuffer pH 8 with EDTA to prevent oxidation of sulfhydryls. Approximately1 pmol Traut's reagent is used to treat 10̂6 cells. Incubate Traut'sreagent with cells for 1 hour at room temperature. Remove excess orunreacted reagent by centrifugation and washing the cells. The number ofavailable sulfhydryl groups can be measured using Ellman's Reagent. Inthe meantime, treat suitable receiver polypeptide withamine-to-sulfhydryl crosslinker, such as SMCC (Pierce) according tomanufacturer's instructions. Excess crosslinking reagent is removed bydesalting. The maleimide-functionalized protein is then incubated withthe thiolated cells for several hours. Unreacted protein is separatedfrom the conjugated cells by centrifugation and washing.

8. Polypeptide—Non-Covalent Surface Attachment

The gene for an antibody scFv against hepatitis B surface antigen (scFv,described in Bose 2003, Molecular Immunology 40:617) is fused to a6-histidine (SEQ ID NO: 27) affinity tag and to the gene encoding thepolypeptide sequence that binds mouse glycophorin A,HWMVLPWLPGTLDGGSGCRG (SEQ ID NO: 28), in a mammalian expression vector(Genlantis). The full fusion protein is produced by transienttransfection of HEK-293T cells using standard methods and purified on aNi-NTA affinity resin (Pierce) according to manufacturer's instructions.The purified fusion protein is incubated with mouse erythrocytes at >100nM concentration to allow for rapid equilibration and binding of thepeptide to glycophorin A.

9. Polypeptide—Lipid Insertion into Membrane

Traut's reagent (Thermo Fisher) is used to generate sulfhydryl groups onan amine-containing suitable receiver polypeptide molecule followingmanufacturer's protocol. The reaction mixture is incubated for 1 h atroom temperature (RT) on a shaker and washed through a spin desaltingcolumn (Zeba, MWCO 7K, Thermo Scientific) following the manufacturer'sinstructions to remove the unreacted Traut's reagent. The generation ofsulfhydryl groups on the modified polypeptide is quantified usingEllman's Reagent (Pierce) based on the manufacturer's protocol.

DSPE-PEG₃₄₀₀-mal (1×10̂−3 M in PBS, 4 μL, molar ratiolipid:Polypeptide=1:1) (all lipids purchased from Avanti Polar Lipidsand stored as chloroform solution under argon at −20 C) are added to thedesalted polypeptide solution and incubated at RT on a shaker. After 1h, the sample solution is filtered using a centrifugal filter device(Microcon, Millipore Co.) at 14 000 g for 15 min at 4° C. to remove thesmall molecules and suspended in 600 μL PBS (1 mg/mL polypeptide).

200 μL of whole blood is suspended in 1000 μL PBS and spun at 1500 g for30 s, repeated four times. Finally, the RBCs are suspended in 800 μLPBS. The conjugation of RBC/DSPE-PEG-Polypeptide is prepared by mixingthe above RBCs suspensions and various amounts of DSPE-PEG-Polypeptidesolution (1 mg per mL) followed by incubation for 15-30 min at 37° C.The mixture is kept for 5 min at room temperature, then washed threetimes in PBS and resuspended to a final RBC concentration of 5×10̂8 permL. An automated cell counter (Countess, Invitrogen) is used to measurethe cell concentration.

10. Polypeptide—Hypotonic Loading

A suitable receiver polypeptide, in this instance mouse IgG, waspurchased from Abcam and was added at 0.25 mg/mL to a RBC suspension inisotonic solution at a hematocrit (Hct) of 70%. The suspension wasdialyzed in 250 mL of a hypotonic solution containing 10 mM sodiumphosphate pH 7.4, 10 mM sodium bicarbonate, and 20 mM glucose, stirredat 15 rpm for 1 hour at 4 C. The cells were then isotonically resealedby adding 1/10 volume of resealing solution comprising 5 mM adenine, 100mM inosine, 100 mM sodium pyruvate, 100 mM sodium phosphate, 100 mMglucose, 12% (w/v) NaCl at pH 7.4. Cells were then incubated at 37 C for30 minutes.

11. Polypeptide—Cell-Penetrating Peptide

The manufacture of protamine-conjugated polypeptide is known in the art,see e.g., Kwon et al. 2009 J Contr Rel 139(3):182.5 mg/ml of LowMolecular Weight Protamine (LMWP) in 50 mM HEPES buffer (pH 8) is mixedwith the heterobifunctional cross-linker 3-(2-pyridyldithio)propionicacid N-hydroxysuccinimide (SPDP, Sigma-Aldrich) at a 1:10 molar ratio inDMSO and shaken for 1 h at room temperature. The reaction mixture isthen treated with 50 mM dithiothreitol (DTT, Sigma-Aldrich) and thethiolated LMWP is purified by HPLC on a heparin affinity column. Theproduct is collected by ultrafiltration, lyophilized, and stored at −20°C. until further use.

For conjugation, 5 mg/ml suitable receiver polypeptide is mixed withSPDP (40 μl of 0.1 M SPDP in ethanol to 1 ml protein solution) inphosphate buffer, and stirred at room temperature for 1 h. UnreactedSPDP is removed by rapid desalting and buffer exchange by FPLC with 0.1M phosphate buffer (pH 7.4). Activated polypeptide is then conjugatedwith a 10-fold molar excess of the above-prepared LMWP-SH for 24 h at 4°C. The LMWP-polypeptide conjugates are isolated by ion-exchangechromatography using a heparin affinity column followed by five roundsof centrifugal filtration (molecular weight cut-off: 5,000 Da). PooledLMWP-polypeptide conjugates are concentrated, and the degree ofconjugation determined by MALDI-TOF mass spectroscopy.

For uptake experiments, fresh sheep erythrocytes (MP Biomedicals, Solon,Ohio) are suspended in Hank's balanced salt solution (HBSS) at a densityof 5×10̂8 cells/ml, and are then incubated with a 0.5 mg/ml solution ofthe LMWP-polypeptide conjugates for 30 min at room temperature undergentle shaking. RBCs are then washed with HBSS and stored at 2-8 C.

12. Polypeptide—Chemical Permeability

3×10̂8 RBCs were preincubated for 30 min with chlorpromazine (SigmaAldrich) at 200 μM in Ringer's solution. Afterwards, the suitablereceiver polypeptide was added in Ringer's solution (1 to 4 μM) to afinal volume of 400 μl and incubated for 30 min at room temperatureunder mild agitation. After incubation, cells were washed twice,resuspended in Ringer and collected for analysis.

13. Polypeptide—Enzymatic Conjugation

Cell surface enzymatic conjugations with sortase are known in the art,see e.g., Shi et al PNAS 2014 111(28):10131. To label the GPA N terminuswith polypeptide, 30 uL of 500 uM S aureus sortase and 1 mM polypeptidewith LPETGG (SEQ ID NO: 29) at the C terminus is preincubated in 50 mMTris pH 7.5, 150 mM NaCl, on ice for 15 minutes and added to 5×10̂7 RBCsin DMEM. The sortase and cell mixture is incubated on ice for 30 minwith occasional gentle mixing, then spun at 500×g for 2 min at 4 C toremove buffer/DMEM, then washed three times with 1 mL of ice-cold PBS.

14. Gas

The following steps are taken to load erythroid cells with nitric oxide(NO). To avoid oxidative side reactions or S-nitrosylation oferythrocytic proteins other than Hb by S-nitrocysteine (CSNO),S-nitrosothiol (SNO)Hb is synthesized in intact RBCs by (i) addition ofaqueous NO to fully deoxygenated RBCs to yield Fe-nitrosylHb[HbFe(II)NO]; (ii) washing under anaerobic conditions; and (iii)reoxygenation, effecting intraerythrocytic intramolecular transfer of NOfrom heme [Fe(II)NO] to Cys-B93. Sulfanilamide [SA; 3.4% (wt_vol)] in0.4MHCl is prepared with and without 1% (wt/vol) HgCl2, as is 0.1%(wt/vol) of N-(1-naphthyl)ethylenediamine (NED). Equal volumes of SNOHbare added to SA with or without HgCl2 and then reacted with NED. [SNO]is determined from the difference in absorbance (540 nm) usingcolorimetry.

15. Small Molecule (Cytoplasm)

Liposomal ProJect reagent (Pierce) is dried under nitrogen into a thinfilm in glass scintillation vials. Approximately 2 uL reagent is neededper 10̂5 cells. Solution of small molecule of interest in PBS is added tothe dried liposome reagent. The solution is pipetted several times,incubated for 5 minutes at room temperature, then vortexed vigorously togenerate encapsulating liposomes. Serum-free medium is added to bringthe total volume to 500 uL per 10̂5 cells. The liposomal mixture isincubated with the cells for 3-4 hours at 37° C.

16. Small Molecule (Surface)

The conjugation of small molecules to the surface of cells usingchemical functionalities is well known in the art, see e.g., Hermanson GT, Bioconjugation Techniques 2^(nd) Ed, ISBN 978-0123705013. Briefly,the small molecule of interest is provided with an amine-reactivefunctional group, such as NHS ester, for example NHS ester biotin(Pierce). The small molecule of interest is stored in organic solvent toprevent hydrolysis of the NHS ester functional group. The small moleculeof interest is incubated with cells in aqueous medium in large molarexcess (at least 10 pmol for 10̂6 cells) to drive conjugation to primaryamines on the cell surface. After 1 hr incubation, the excess unreactedmolecule is removed by centrifugation and washing of the cells.

Example 8: Assessment of Polypeptide Presence

1. Fluorescent Transgene

Erythroid cells were cultured as described herein. A transgene encodingglycophorin A with an HA tag on the C-terminus fused to GFP with anintervening viral T2A peptide was constructed by Gibson assembly asdescribed herein. The transgene was introduced into the erythroid cellsby lentiviral transduction as described herein. Two days aftertransduction, cells were collected, washed in PBS buffer, and analyzedon a flow cytometer (Attune, Life Technologies). Transduction efficiencywas assessed as the percentage of GFP-positive cells in the population.

2. Cell Surface Proteins

For cell surface proteins, the level of protein expression can bedetected as early as 2 days after transfection by flow cytometry withantibodies specific for the protein or for a co-expressed epitope tag.Erythroid cells were cultured as described herein. A transgene encodingglycophorin A with an HA tag at the N-terminus was constructed by Gibsonassembly as described herein. The transgene was introduced into theerythroid cells by lentiviral transduction as described herein. Two daysafter transduction, cells were collected, washed in PBS buffer, andstained with 1:50 dilution of mouse anti-HA antibody (Abcam) for 1 hr.Cells were washed and then stained with a 1:100 dilution of alexa488-labeled goat anti-mouse secondary antibody (Life Technologies) for30 minutes on ice. Cells were washed and analyzed on a flow cytometer(Attune, Life Technologies). Transduction efficiency was assessed as thepercentage of alexa 488-positive cells in the population.

3. Intracellular Proteins

For intracellular proteins, the level of protein expression can bedetected as early as 8-12 hours after transfection by Western Blot.Erythroid cells were cultured as described herein. A transgene encodingadenosine deaminase with an HA tag at the C-terminus was constructed byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Twodays after transduction, cells were collected, washed in PBS buffer, andlysed in RIPA cell lysis buffer (Pierce). Cell lysate was denatured byboiling in 100 mM DTT, then loaded onto a NuPage SDS-PAGE pre-cast gel.After electrophoresis and transfer to nitrocellulose membrane, proteinbands were developed by staining with 1:5000 dilution of mouse anti-HAantibody (Abcam) followed by 1:5000 dilution of goat anti-mouse HRP(Pierce), and subsequent treatment with HRP substrate (SuperSignal,Pierce). Images were captured using an Amersham imager (GE healthcare).

Example 9: Assessment of Small Molecule Presence

Eyrthrocytes from a normal human donor were purchased (Research BloodComponents). Cells were then biotinylated with NHS-biotin (Sigma) permanufacturer's instructions using 0.02× volumes of 2 mM stock biotinreagent for 30 minutes at room temperature. Anti-biotin antibody (Abcam)was fluorescently labeled with Dylight 650 (Pierce). Labeling efficiencyof the cells was assessed by flow cytometry as described herein usingthe labeled anti-biotin antibody as a detection marker.

Example 10: Assessment of Gas Level

A standard protocol is used to determine NO2- and NO3-levels in thethree blood components, see e.g., Yang et al. 2003, Free Radic Res37(1):1. Briefly, a “stop solution” (K3Fe(CN)6, N-ethylmaleimide, water,NP40) is added to blood to maintain nitrite levels until sampleanalysis. A 1:4 dilution of “stop solution” to blood is vortexed andplaced on dry ice. At the time of sample analysis, a 1:1 dilution of99.9% pure methanol and thawed sample is centrifuged for 2 min at 13,000rpm; the supernatant is immediately injected into the chemiluminescentnitric oxide analyzer (NOA, Sievers, Model 280 NO analyzer, Boulder,Colo.) using helium as the carrier gas. The triiodide (I3-) ozone-basedchemiluminescent assay is used to analyze nitrite levels. To analyzenitrate, deionized water (Millipore CQ-Gard, Bedford, Mass.) is added toblood to lyse cells. A 9:1 dilution of deionized water to blood isvortexed and placed on dry ice. At the time of sample analysis, a 3:1dilution of pure HPLC grade ethanol and thawed sample is centrifuged,and the supernatant is immediately analyzed using aVanadium(III)chloride chemiluminescent assay, see e.g., Ewing andJanero, 1998 Free Radic Biol Med 25(4-5):621. The VC13 reaction solutionis maintained at 90° C. with helium as the carrier gas. 1 μM nitrite andnitrate solutions are used to generate standard curves for comparisonsand adjustments of sample nitrite and nitrate concentrations.

A thiol-stabilization solution (NEM-DPTA; K3Fe(CN)6, N-ethylmaleimide,Diethylenetriaminepenta acetic acid, NP40, water) is added to blood tomaintain SNOHb and HbNO levels by inhibiting additional thiol reactions.A 4:1 dilution of NEM-DPTA to blood is vortexed and placed on dry ice. A9:1 dilution of sample and 5% acid sulfanilamide (AS) is incubated for 5min; half is injected into the NOA (I3-assay) to give combined SNOHb andHbNO levels. The remaining sample is incubated with 50 mM HgCl2, thenincubated again with 5% AS, and injected into the NOA to give HbNOlevels.

Example 11: Assessment of Expression and Activity

The expression of exogenous proteins in and on cultured cells can beassessed quantitatively by flow cytometry (if the protein is expressedon the surface) or by Western blot (for proteins expressed in thecytoplasm).

1. Quantitative Flow Cytometry

Anti-mouse Fc-binding quantitative flow cytometry beads (Simply CellularCalibration) were purchased from Bangs Labs. Fluorescently labeled mouseantibodies against relevant cell surface receptors—glycophorin A, Ckit,and transferrin receptor—were purchased from BioLegend. Fluorescentlylabeled mouse antibody against the HA epitope tag was purchased fromLife Technologies. Erythroid cells were cultured as described herein. Atransgene encoding glycophorin A with an HA tag at the N-terminus wasconstructed by Gibson assembly as described herein. The transgene wasintroduced into the erythroid cells by lentiviral transduction asdescribed herein. At least two days after transduction, 2×10̂5 cells werecollected, washed in PBS buffer, and stained with 1:100 dilution of oneof the above-listed antibodies for 1 hr. Cells were washed and analyzedon a flow cytometer (Attune, Life Technologies). The protocol wasrepeated for each of the four antibodies listed above. Quantificationwas performed according to manufacturer's instructions. Briefly, onedrop of each of the five bead samples was incubated with 1:100 dilutionof an above-listed antibody. The beads were incubated for 1 hr, washedin PBS, and analyzed on a flow cytometer (Attune, Life Technologies).The protocol was repeated for each of the four antibodies listed above.Calibration curves were fit using the manufacturer's provided excelspreadsheets, from which quantification of fluorescence intensity forthe cell-based signals was derived.

2. Quantitative Western Blot

Erythroid cells were cultured as described herein. A transgene encodingadenosine deaminase with an HA tag at the C-terminus was constructed byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Twodays after transduction, cells were collected, washed in PBS buffer, andlysed in RIPA cell lysis buffer (Pierce).

The transgene was introduced into HEK293T cells by transienttransfection using lipofectamine 2000 (Life technologies). Cells werecultured for one week and the supernatant was harvested. Recombinantprotein was purified on an HA affinity column (Pierce) according tomanufacturer's instructions. Protein concentration was assessed byabsorbance at 280 nm.

Western blotting was performed as described herein. In addition to thecell lysate samples, known amounts of the recombinant adenosinedeaminase were run on the same gel. Following image collection, theintensity of the recombinant bands were used to generate a standardcurve to quantify the amount of protein present in the cell samples.

The robust expression of transgenes at high levels has importantimplications for the therapeutic capacity of the final cell population.FIG. 2 quantifies the expression of three surface proteins indicative ofdifferentiation and one exogenous transgene by quantitative flowcytometry, and demonstrates that the transgene is robustly expressed ata high level.

Erythroid cells in culture were collected at seven time points during afour-stage in vitro differentiation process. At the first time point(“Expand D6”) the cells are nucleated hematopoietic precursors. By thefinal time point (“Diff 3 D8”) the cells are predominantly enucleatederythroid cells. GPA (solid triangles), a canonical marker of erythroidcells, starts low in the precursor cells and rapidly reaches >1×10̂6copies per cell. CKIT (dashed squares), a receptor for stem cell factor,starts high then decreases to <1×10̂4 copies per cell as differentiationensues. TR (dotted diamonds), necessary for the transport of iron intoerythroid cells, increases initially then gradually declines to <1×10̂5copies per cell. The transgene (open circles) is introduced at the endof the second differentiation stage (“Diff 1”) and is steadily expressedat approximately 1×10̂5 copies per cell throughout differentiation. Theabove data demonstrate that transgenes are robustly expressed incultured cells.

The expression of exogenous proteins in and on cultured cells can beassessed by flow cytometry (if the protein is expressed on the surface)as described herein, or by Western blot (for proteins expressed in thecytoplasm) as described herein. In instances where an exogenous gene isin a single-transcript construct that contains a downstream fluorescentreporter protein, the fluorescence of the reporter protein can be usedas a proxy for expression of the upstream gene, and assessed by flowcytometry as described herein.

FIG. 3 A-FIG. 3N shows the exogenous expression of surface andcytoplasmic proteins on enucleated cultured erythroid cells. The abovedata conclusively demonstrate that multiple protein classes—includingcytoplasmic, surface, intact, fusions to type I membrane proteins,fusions to type II membrane proteins, fusions to GPI-linked membraneproteins, intracellular fusions, overexpressed, and de novoexpressed—can be expressed on multiple cell types including culturedenucleated erythroid cells, cultured nucleated erthyroid precursorcells, and K562 erythroleukemia cells.

FIG. 3B and FIG. 3D demonstrate the simultaneous expression of twoexogenous proteins in an enucleated cultured cell.

In FIG. 3B, Erythroid cells were cultured as described herein. Atransgene construct encoding glycophorin A signal sequence, an HAepitope tag, glycophorin A coding sequence, viral T2A cleavable sequenceand GFP was assembled by Gibson assembly as described herein. Thetransgene was introduced into the erythroid cells by lentiviraltransduction as described herein. The cells were cultured to terminaldifferentiation as described herein. Cells were analyzed by flowcytometry as described herein, using a fluorescent anti-HA antibody andGFP fluorescence to detect expression of both transgenes.

In FIG. 3D, Erythroid cells were cultured as described herein. Atransgene construct encoding glycophorin A signal sequence, antibodyscFv specific to hepatitis B surface antigen, HA epitope tag,glycophorin A coding sequence, viral T2A cleavable sequence and GFP wasassembled by Gibson assembly as described herein. The transgene wasintroduced into the erythroid cells by lentiviral transduction asdescribed herein. The cells were cultured to terminal differentiation asdescribed herein. Cells were analyzed by flow cytometry as describedherein, using a fluorescent anti-HA antibody and GFP fluorescence todetect expression of both transgenes.

Example 12: Expression of Protein from mRNA in Platelets

The expression in platelets of exogenous proteins translated fromexogenous transfected mRNA was measured by flow cytometry. In brief,platelet-enriched serum was centrifuged at 190 g for 15 minutes toremove erythrocytes and leukocytes. The supernatant was then spun for anadditional 5 minutes at 2500 g to pellet platelets. Platelets wereresuspended in 5 mL of Tyrode's buffer with 1 uM prostaglandin, washed,and resuspended in 750 uL of Tyrode's buffer with 1 uM prostaglandin.mRNA encoding the gene of interest, in this example GFP, was mixed withlipofectamine at a 1:1 mg/mL ratio. The mixture was incubated for 5minutes, then added to the washed platelet population. The combinationwas incubated for 24 hours at room temperature with slow rocking.Platelet expression of the transgene was assayed by flow cytometrymeasuring GFP fluorescence. Surface proteins can also be assayed by flowcytometry. Cytoplasmic or other intracellularly-expressed proteins canalso be assayed by Western blot.

There is therapeutic relevance to introducing exogenous proteins intoand onto platelets. Since platelets do not possess a nucleus or RNAtranscription machinery, DNA transfection is not a viable means ofinducing exogenous protein expression in platelets. However, mRNAtransfection and translation is a way of introducing exogenous proteinsinto cells. It is thought that platelets contain mRNA translationmachinery, but until now it was not known whether they are able toaccept and translate exogenous mRNA into protein.

FIG. 4A-FIG. 4C is a collection of flow cytometry plots that demonstratethe translation of exogenous mRNA encoding a transgene, in this caseGFP, by platelets. The GFP is detected by fluorescence in the FL1channel after excitation with a 488 nm laser. (FIG. 4A) Untransfectedplatelets (1.7% GFP+). (FIG. 4B) Platelets transfected with 3 ug GFPmRNA (8.6% GFP+). (FIG. 4C) Platelets transfected with 6.8 ug GFP mRNA(3.3% GFP+).

The data conclusively demonstrate, for the first time, the translationof exogenous mRNA into exogenous protein by platelets.

Example 13: Activity of Enzymes

FIG. 5A-FIG. 5D demonstrates the activity for enzymes contained onerythroid cells. Biochemical activity of cytoplasmic enzymes wasassessed by Western blot for retention of a protein over the course ofdifferentiation. Biological activity of cytoplasmic enzymes was assessedby in vitro enzymatic activity assay.

FIG. 5A-FIG. 5D shows the activity of two different intracellularenzymes expressed in cultured erythroid cells.

1. Adenosine Deaminase.

A transgene encoding adenosine deaminase with an HA tag at theC-terminus was constructed by Gibson assembly as described herein. Thetransgene was introduced into HEK-293T cells by lipofectaminetransfection (Life Technologies) as described herein. Enzymatic activityis assayed using a protocol derived from Helenius 2012, Biochim BiophysActa 1823(10):1967, in which a specific mixture of enzymes convertpurines into uric acid and H2O2 followed by fluorometric detection ofthe generated H2O2. In brief, two days after transfection, cells werecollected, media aspirated, and Krebs Ringer phosphate glucose (KRPG;comprising: 145 mM NaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mMCaCl2, 1.22 mM MgSO4, and 5.5 mM glucose; pH 7.35) added to the cells at2×10̂5 cells/mL. Adenosine was added at 50 uM. After reaction for 6hours, supernatant was collected and heat inactivated for 5 minutes at60 C. Aliquots of supernatant were transferred to wells in a white96-well microplate containing 0.25 U/ml bacterial purine nucleosidephosphorylase (PNP) and 0.15 U/ml microbial xanthine oxidase (XO), bothfrom Sigma. After 20 min incubation at RT, 30 μl of H2O2-detectingmixture containing HRP (final concentration 1 U/ml, Sigma) and AmplexRed reagent (60 μM, Invitrogen, Molecular Probes) was added to themicrowells, followed by measurement of the fluorescence intensity at theemission and excitation wavelengths of 545 and 590 nm, respectively(Tecan Infinite M200).

2. Phenylalanine Hydroxylase

Erythroid cells were cultured as described herein. A transgene encodingphenylalanine hydroxylase with an HA tag at the C-terminus wasconstructed by Gibson assembly as described herein. The transgene wasintroduced into the erythroid cells by lentiviral transduction asdescribed herein. Two days after transduction, cells were collected,washed in PBS buffer, and lysed in RIPA cell lysis buffer (Pierce). Celllysates (64 ug total protein) were added to 1 mL reaction buffercontaining 100 mM Tris-HCl, pH 7.5, 4 mM DTT, 4 mM Phenylalanine, 33 μgcatalase, and 0.4 mM DMPH4 (all from Sigma). Reactions were runovernight at 37 C. After incubation, samples were de-proteinized bycentrifugal filtration in an Amicon Ultra-4 Centrifugal Filter 10 KD(Millipore UFC801024) spinning at 3700 rpm for 10 min. Samples werecollected and assayed for tyrosine concentration by absorbance at 540nm.

Both of these exogenous proteins were retained through the end ofterminal differentiation, a non-trivial feat given that it is well-knownin the field that erythroid cells undergo a rigorous program ofelimination of proteins unnecessary for basic function (Liu J et al.(2010) Blood 115(10):2021-2027, Lodish H F et al. (1975) DevelopmentalBiology 47(1):59). In FIG. 5A, the exogenously over-expressed proteinadenosine deaminase is detected by anti-HA Western blot at various timepoints over the course of differentiation, from nucleated precursorcells (“Diff I D5”) through to enucleated erythroid cells (“Diff IIID8”). In FIG. 5C, the exogenously expressed microbial proteinphenylalanine hydroxylase is detected by anti-HA Western blot at varioustime points over the course of differentiation, from nucleated precursorcells (“Diff I D5”) through to enucleated erythroid cells (“Diff IIID8”).

Additionally, both of these enzymes maintained their ability toenzymatically convert substrate into product. FIG. 5B shows theenzymatic conversion of adenosine to inosine by intact adenosinedeaminase-expressing 293T cells. FIG. 5D shows the enzymatic conversionof phenylalanine to tyrosine by lysates of cultured phenylalaninehydroxylase-expressing enucleated erythroid cells.

These data conclusively demonstrate that exogenous enzymes are retainedon erythroid cells throughout the culture process and that they areenzymatically active in erythroid cells, which has profound therapeuticimplications.

Example 14: Activity of CR1

FIG. 6A-FIG. 6B shows both biochemical and biological activity forcomplement receptor 1 (CR1) over-expressed on the surface of culturederythroid cells. Biochemical activity of CR1 was assessed by flowcytometry for binding to an immune complex. Biological activity of CR1was assessed by transfer of immune complexes to macrophages in aco-culture assay.

1. Immune Complex Binding of CR1-Expressing Cells.

Erythroid cells were cultured as described herein. A transgene constructencoding complement receptor 1 (CR1) was constructed by Gibson assemblyas described herein. The transgene was introduced into the erythroidcells by lentiviral transduction as described herein. Transgeneexpression levels were assessed by flow cytometry as described hereinusing an anti-CR1 antibody (Abcam). The cells were cultured to terminaldifferentiation as described herein.

Dylight 650-labeled bovine serum albumin (BSA-650) was incubated withpolyclonal rabbit anti-BSA (Abcam) in an excess of antibody for 30minutes at room temp. The complexes were then mixed with human serum ata 1:1 volume ratio for 30 minutes at 37 C. Control complexes were eithernot mixed with human serum or mixed with heat-inactivated human serum.

Complexes were incubated with the CR1-expressing cells for 30 minutes at37 C. Cells were washed and analyzed by flow cytometry for capture ofimmune complexes by detecting Dylight 650 fluorescence.

2 Immune Complex Transfer to Macrophages

Cultured U937 monocytes were activated by incubation with 100 nM phorbolmyristate acetate (PMA) for 24 hours at 37 C. Cells coated with immunecomplexes (see above) were incubated with activated U937 macrophages for30 minutes at 37 C. The co-culture was analyzed by flow cytometry.Macrophages were identified by FSC/SSC gating. Presence of immunecomplex on macrophages was analyzed by detecting Dylight 650fluorescence in the macrophage population.

FIG. 6A-FIG. 6B shows the biochemical and biological activity ofcomplement receptor 1 (CR1) exogenously over-expressed on culturederythroid cells.

FIG. 6A shows the biochemical activity of CR1, defined as the capture ofimmune complexes in vitro. The black histogram shows the capture ofBSA-based immune complexes by CR1 over-expressed on cultured erythroidcells. The shaded histogram shows the minimal background binding tocomplexes of BSA and IgG that lack human complement, demonstrating thatthe binding event is CR1-mediated.

FIG. 6B shows the biological activity of CR1, defined as the transfer ofcaptured immune complexes from cultured erythroid cells to macrophages.This is a standard assay in the field, see: Repik et al. 2005 Clin ExpImmunol. 140:230; Li et al. 2010 Infection Immunity 78(7):3129. Transferis assessed by flow cytometry and measured as the intensity of labeledimmune complex-derived fluorescence on macrophages. In this assay,macrophages that are incubated with no immune complexes (black bars) donot become fluorescent. Macrophages that are incubated with complexes ofBSA and IgG that lack complement (and therefore do not bind CR1) take uponly a small amount of immune complex (solid gray bars), independent ofthe presence of cultured CR1-overexpressing erythroid cells. This uptakeis likely due to Fc-gamma receptors on the U937 cells interacting withthe Fc regions of the IgG molecules. Macrophages that are incubated withimmune complexes (BSA+IgG+complement) in the absence ofCR1-overexpressing cells (hashed bar, left) take up the same amount ofimmune complex as in the absence of complement, likely by the sameFc-gamma mediated method. However, the macrophages that are incubatedwith immune complexes in the presence of CR1-overexpressing cells(hashed bar, right) take up nearly double the number of immune complexesas measured by fluorescence.

These data conclusively demonstrate that CR1 overexpression on culturederythroid cells enables the capture of immune complexes on saiderythroid cells, facilitates the transfer of immune complexes fromerythoroid cells to macrophages, and significantly increases the rateand number of immune complexes taken up by macrophages.

Example 15: Activity of scFv

FIG. 7A-FIG. 7D shows the biochemical and biological activity ofantibody scFv exogenously expressed on the surface of cultured erythroidcells as a fusion to the transmembrane protein GPA.

Erythroid cells were cultured as described herein. A transgene constructencoding the leader sequence of glycophorin A, an antibody scFv specificto hepatitis B surface antigen (scFv, described in Bose 2003, MolecularImmunology 40:617), an HA epitope tag, a [Gly-3-Ser]2 flexible linker,and the body of glycophorin A was assembled by Gibson assembly asdescribed herein. The transgene was introduced into the erythroid cellsby lentiviral transduction as described herein. Transgene expression wasassessed by flow cytometry as described herein using an anti-HA antibody(Abcam). The cells were cultured to terminal differentiation asdescribed herein. Biochemical activity of the antibody scFv was assessedby flow cytometry for binding to the target protein, in this casehepatitis B surface antigen (HBsAg). Recombinant HBsAg protein (Abcam)was labeled with Dylight-650 fluorophore (Pierce). scFv-expressing cellswere incubated with 100 nM labeled protein, washed in PBS, and analyzedfor Dylight 650 fluorescence by flow cytometry as described herein.

Biological activity of the antibody scFv was assessed by in vivo captureof HBsAg detected by flow cytometry. Recombinant HBsAg protein (Abcam)was labeled with Dylight-650 fluorophore (Pierce). scFv-expressing cellswere fluorescently labeled with CFSE (Sigma) Immunocompromised NSG mice(Jackson labs) were injected with ˜400 pmol of the labeled HBsAg intothe tail vein. A few minutes later, the same mice were injected with2×10̂7 scFv-expressing cells. Blood was collected by submandibularpuncture at regular intervals in an EDTA-containing tube. Collectedblood cells were washed and analyzed by flow cytometry as describedherein. Human cells were identified as those that were CFSE positive.Capture of HBsAg was detected as Dylight 650 fluorescence on the humancells.

FIG. 7A-FIG. 7B show the biochemical activity of antibody scFv, definedas the binding of its cognate antigen, hepatitis B surface antigen(HBsAg). In FIG. 7A, cells that express (black) or do not express (grayshaded) the antibody scFv are incubated with 450 nM HBsAg and stainedwith biotinylated anti-HBsAg antibody and fluorescent streptavidin.Cells that express the antibody scFv (45% of the cells in this culture)bind to the antigen. In FIG. 7B, cells that express the antibody scFvare incubated with various concentrations of HBsAg and stained as above,showing that the binding event is dose-dependent with an affinity ofapproximately 10 nM.

FIG. 7C-FIG. 7D show the biological activity of antibody scFv, definedas the capture of cognate antigen HBsAg while in circulation in a mouse.In this experiment, immunocompromised NSG mice were injected with ˜400pmol fluorescently-labeled HBsAg via the tail vein. Five minutes later,cultured enucleated erythroid cells (7C) or cultured enucleatederythroid cells that expressed exogenous antibody scFv (7D) wereinjected via the tail vein. Prior to injection, all cultured cells werelabeled with CFSE fluorescent dye. Blood was collected 6 hours later,analyzed on a flow cytometer, and gated on CFSE+ human cells. Barecultured cells did not bind to HBsAg (7C), whereas antibodyscFv-expressing cells do bind to HBsAg (7D). Consistently with thebiochemical activity experiment, approximately 45% of the cells in thisculture express antibody-scFv.

These data demonstrate that the antibody scFv is biochemically activewhen expressed on the surface of cultured erythroid cells and that theantibody scFv on the erythroid cell is able to bind its target in vivowhen in circulation. This has profound implications for therapeuticapproaches in which the capture, sequestration, and clearance of asubstance in circulation is desired.

Example 16: Activity—Circulating Clearance

FIG. 8A-FIG. 8D shows both biochemical and biological activity forsurface molecule capture agents used for circulating clearance of atarget.

Biochemical activity of the capture agents, in this case HA polypeptideand biotin, was assessed by flow cytometry for binding to the targetprotein, in this case anti-HA antibody and anti-biotin antibody.Biological activity of the capture agents was assessed by in vivocapture and clearance of target protein as detected by flow cytometryand plasma protein quantification.

1. Capture of Anti-Biotin Antibody by Chemically-Modified Cells

Eyrthrocytes from a normal human donor were purchased (Research BloodComponents). Cells were labeled with CFSE (Sigma) per manufacturer'sinstructions for 20 minutes at 37 C. Cells were then biotinylated withNHS-biotin (Sigma) per manufacturer's instructions using 0.02 volumes of2 mM stock biotin reagent for 30 minutes at room temperature.Anti-biotin antibody (Abcam) was fluorescently labeled with Dylight 650(Pierce). Labeling efficiency of the cells was assessed by flowcytometry using the labeled anti-biotin antibody and CFSE fluorescenceas detection markers. 250 ug labeled antibody was injected into an NSGmouse (Jackson Labs) intravenously via the tail vein. Four hours later1×10̂8 biotinylated cells were injected intravenously via the tail vein.Blood was collected by submandibular puncture at regular intervals in anEDTA-containing tube. Collected blood cells were washed and analyzed byflow cytometry as described herein. Human cells were identified as thosethat were CFSE positive. Capture of anti-biotin antibody was detected asDylight 650 fluorescence on the human cells. Plasma from the blood drawwas analyzed by ELISA using a biotin-coated microplate (Pierce) permanufacturer's instructions to detect the level of antibody incirculation.

2. Capture of Anti-HA Antibody by Transgenic Cultured Cells

Erythroid cells were cultured as described herein. A transgene constructencoding glycophorin A signal sequence, an HA epitope tag, glycophorin Acoding sequence, viral T2A cleavable sequence and GFP was assembled byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Thecells were cultured to terminal differentiation as described herein.Cells were analyzed by flow cytometry as described herein, using ananti-HA antibody (Life Technologies) fluorescently labeled with Dylight650 (Pierce) and GFP fluorescence to detect expression of bothtransgenes. 250 ug labeled anti-HA antibody was injected into an NSGmouse (Jackson Labs) intravenously via the tail vein. Four hours later1×10̂8 cultured cells were injected intravenously via the tail vein.Blood was collected by submandibular puncture at regular intervals in anEDTA-containing tube. Collected blood cells were washed and analyzed byflow cytometry as described herein. Human cells were identified as thosethat were CFSE positive. Capture of anti-HA antibody was detected asDylight 650 fluorescence on the human cells. Plasma from the blood drawwas analyzed by ELISA using an HA peptide-coated microplate (Pierce) permanufacturer's instructions to detect the level of antibody incirculation.

FIG. 8A-FIG. 8D shows biochemical and biological activity of (FIG.8A-FIG. 8B) the polypeptide HA expressed on the surface of culturederythroid cells as a fusion to GPA and of (FIG. 8C-FIG. 8D) biotinchemically conjugated to the surface of primary erythrocytes.Biochemical activity is defined as the capture of a target protein invitro. Biological activity is defined as the enhanced clearance of atarget protein in vitro.

In FIG. 8A, the HA polypeptide, expressed as a fusion to the N terminusof GPA, captures a mouse anti-HA antibody in vivo. NSG mice wereinjected with fluorescently-labeled mouse anti-HA antibody, followed byinjection of cultured human erythroid cells that either do not (left) ordo (right) express HA epitope tag on their surface as a fusion to GPA.Blood was drawn and cells analyzed on the flow cytometer. The x-axismeasures CFSE fluorescence. The y-axis measures anti-HA antibody Dylight650 fluorescence. CFSE-positive cultured human erythrocytes are observedin both samples, but only the cells expressing the HA epitope tag areable to capture circulating anti-HA antibody.

In FIG. 8B, mice were injected with anti-HA antibody then thenoptionally with cultured human erythroid cells that either do not or doexpress HA peptide on their surface as a fusion to GPA. Plasma wascollected at multiple time points and the level of anti-HA antibody inplasma was assessed by ELISA using an HA peptide-coated plate as asubstrate. Mice injected with anti-HA antibody alone (open circles,solid line—this mouse died after 120 minutes of causes unrelated totreatment) or with anti-HA antibody followed by cells that do notexpress HA peptide on their surface (dashed line) have significantantibody in circulation out to 24 hours post injection of cells. Incontrast, mice injected with anti-HA antibody followed by cells thatexpress HA peptide on their surface are depleted of target antibodywithin minutes. This data conclusively demonstrates that the targetantibody is rapidly and specifically cleared from circulation bycultured erythroid cells that express receiver polypeptide on theirsurface.

In FIG. 8C, the biotin molecule, conjugated to the surface of erythroidcells by amine functionalization chemistry, captures a mouse anti-biotinantibody. In both of these cases capture was assessed by flow cytometry.Cells that are CFSE labeled and biotinylated show up as double positivewhen stained with a fluorescent anti-biotin antibody (lower dot plot),whereas CFSE-labeled cells that are not biotinylated only show up assingle positive (upper dot plot).

In FIG. 8D, mice were injected with anti-biotin antibody then thenoptionally with cultured human erythroid cells that either are not orare conjugated to biotin on their surface. Plasma was collected atmultiple time points and the level of anti-biotin antibody in plasma wasassessed by ELISA using a biotin-coated plate as a substrate. Miceinjected with anti-biotin antibody alone (open circles, solid line) orwith anti-biotin antibody followed by cells that are not conjugated tobiotin on their surface (dashed line) have significant antibody incirculation out to 24 hours post injection of cells. In contrast, miceinjected with anti-biotin antibody followed by cells that are conjugatedto biotin on their surface are depleted of target antibody withinminutes. This data conclusively demonstrates that target antibodies arerapidly and specifically cleared from circulation by cultured erythroidcells that contain receiver polypeptide on their surface.

Together the data conclusively demonstrate that suitable receivers onmembrane-receiver complexes are able to bind their target molecules invivo and mediate rapid circulating clearance of target molecules when incirculation, which has profound therapeutic implications.

Example 17: Activity of Complement Regulators

The complement regulatory activity of the synthetic membrane-receivercomplexes is assessed by standard CH50 and AH50 assays known in the art(see e.g., Kabat et al. 1961 Exp Immunochem pp. 133-239 and Platts-Millset al. 1974 J Immunol 113:348).

Briefly, the CH50 assay utilizes sheep erythrocytes (SRBC) as targetcells. Briefly, a suspension containing 1×10̂9 SRBC/ml is prepared in theGVB(2+) buffer (gelatin/Veronal-buffered saline with Ca2+ and Mg2+), pH7.35. Hemolysin (rabbit anti-sheep antiserum) is titrated to determinethe optimal dilution to sensitize SRBC. Diluted hemolysin (1:800) ismixed with an equal volume of SRBC (1×109 SRBC/ml), and the whole isincubated at 37° C. for 15 minutes. This results in 5×10̂8/mlantibody-coated erythrocytes (EA). EA (100 μl) are incubated with 100 μlof five serial twofold dilutions (1:20, 1:40, 1:80, 1:160, and 1:320) ofthe normal human serum (NHS) or similar dilution of the mixture of NHSand the membrane-receiver complex at 37° C. for 1 hour. NHS incubatedwith GVB2+ buffer is used as the control. Background control is obtainedby incubating EA with buffer alone (serum is not added), and total lysis(100% hemolysis) is determined by adding distilled water to EA. Thereaction is stopped using 1.2 ml of ice-cold 0.15 M NaCl, the mixture isspun to pellet the unlysed cells, and the optical density of thesupernatant is determined spectrophotometrically (412 nm). Thepercentage of hemolysis is determined relative to the 100% lysiscontrol. Complement activity is quantitated by determining the serumdilution required to lyse 50% of cells in the assay mixture. The resultsare expressed as the reciprocal of this dilution in CH50 units/ml ofserum.

Briefly, the AH50 assay depends on lysis of unsensitized rabbiterythrocytes (Erab) by human serum by activation of the alternativepathway. Activation of the calcium-dependent classical pathway isprevented by addition of the calcium chelator ethylene glycoltetraacetic acid (EGTA) to the assay buffer, and magnesium, necessaryfor both pathways, is added to the buffer. Briefly, a cell suspension ofrabbit RBC (2×10̂8 cell/ml) is prepared in the GVB-Mg2+-EGTA buffer. Aserial 1.5-fold dilution (1:4, 1:6, 1:9, 1:13.5, and 1:20.25) of normalhuman serum (NHS) or similar dilution of the mixture of NHS and themembrane-receiver complex are prepared in GVB-Mg2+-EGTA buffer, and 100μl of each serum dilution is added to 50 μl of standardized Erab. NHSincubated with GVB-Mg2+-EGTA buffer is used as the control. The mixtureis then incubated at 60 minutes at 37° C. in a shaking water bath tokeep cells in suspension, and 1.2 ml of ice-cold NaCl (0.15 M) is usedto stop the reaction. The tubes are spun at 1250 g, at 4° C., for 10minutes to pellet the cells, and the optical density of the supernatantis determined spectrophotometrically (412 nm). Background control has100 μl GVB-Mg2+-EGTA buffer, and 50 μl Erab and does not exceed 10% ofthe total lysis. In the total lysis control tube 100 μl of distilledwater is added to 50 μl Erab suspension, and the percentage of hemolysisis determined relative to 100% lysis control. The results of the assayare calculated and complement activity is quantitated by determining theserum dilution required to lyse 50% of cells in the assay mixture. Theresults are expressed as the reciprocal of this dilution in AH50units/ml of serum.

Example 18: Activity of Platelet-Loaded Thymidine Phosphorylase

A transgene encoding thymidine phosphorylase with an HA tag at theC-terminus is constructed by Gibson assembly as described herein.Platelets are cultured from precursor cells as described herein. Thetransgene is introduced into the cultured platelet precursor cells bylentiviral transduction as described herein. Expression of thymidinephosphorylase within the cultured platelets is assessed by Westernblotting using an anti-HA detection antibody, as described herein.

Thymidine phosphorylase activity is determined in platelet samples byquantifying the rate of conversion of thymidine to thymine. Preliminaryexperiments are conducted to determine the linear metabolite formationkinetics with respect to time and enzyme dilution; the method is shownto be linear for up to 16 min, over a thymine phosphorylase range of4.0-719 nmol/min/ml (corresponding to a sample dilution range of10-9088). Lysates of pre-dialysis samples cultured platelet and controlplatelet samples are prepared by diluting thawed samples 1:710 with 125mM Tris-HCl, pH 7.4. Twenty-five ul of the platelet lysate is then addedto 100 ul sodium phosphate buffer (100 mM, pH 6.5) and 25 ul thymidinestandard (10 mM), mixed and incubated at 37 C for 10 min. The reactionis terminated with 25 ul of 40% TCA. Assay blanks are prepared by addingTCA to the sodium phosphate buffer/thymidine incubation mixture prior toadding the platelet lysate. Samples are centrifuged at 13,400×g for 2min, and the supernatant washed twice with water-saturated diethyl etherwith 2 min on a shaker to extract the TCA. To avoid ether interferingwith HPLC separation, effective removal is achieved by exposing thematrix to the air for 5 min to allow evaporation of the ether. A samplevolume of 10 ul is injected into the HPLC.

Chromatographic separation of substrate and product is achieved usingreversed phase chromatography with isocratic elution using a WatersAlliance HPLC 2795 system. A pre-packed C18column (Spherisorb ODS 125mm×4.6 mm ID, 5 um particle size, Waters) is used as the stationarystage. Analytes are eluted using a mobile phase of ammonium acetate (40mM) with the ion-pairing agent tetrabutyl ammonium hydrogen sulphate (5mM) adjusted to pH 2.70 with HCl, delivered at a flow rate of 1.0ml/min, with a run time of 8 min. UV detection is at 254 nm and 0.1absorbance units full scale. Metabolites are identified by comparingspectra with pure standards.

Example 19: Activity of Platelet-Displayed Goodpasture Antigen

A transgene encoding collagen alpha-3(IV) (COL4A3) NC1 domain antigenfused to the N terminus of CD42b (GP1B, genbank AAH27955.1) with anintervening HA tag is constructed by Gibson assembly as describedherein. Platelets are cultured from precursor cells as described herein.The transgene is introduced into the cultured platelet precursor cellsby lentiviral transduction as described herein. Expression of theexogenous antigen on the cultured platelets is assessed by flowcytometry using an anti-HA detection antibody as described herein.

Serum is collected from a patient suffering from Goodpasture's syndrome,and the serum is tested for anti-COL4A3 antibodies by commercial ELISA(MyBioSource COL4A3 ELISA Kit). The binding capacity of theantigen-expressing platelets is assessed by flow cytometry as describedherein, using this anti-COL4A3 serum as the primary detection antibodyand fluorescent anti-human IgG as the secondary detection antibody.

Platelet-facilitated clearance of a circulating antigen in vivo ismodeled in a mouse using the antigen-expressing platelets. NSG mice areinjected with 100 uL of mouse anti-human COL4A3 antibody (CreativeBioMart) fluorescently labeled with Dylight 650 dye. CFSE-labeledcultured platelets (10̂8 per mouse) that express the exogenous antigenare then injected via the tail vein. Blood is drawn from a submandibularlocation at 10 min, 30 min, 2 h, 12 h, and 24 h. Blood is centrifuged tocollect the platelet-rich fraction, which is then stained and analyzedby flow cytometry as described herein. Antibody capture by platelets isdetermined by tracking the CFSE-Dylight 650 double positive population.

Example 20: Activity In Vivo (Mouse)

Mouse erythroid cells are cultured as described herein. Erythroidprecursor cells are transduced with a suitable receiver polypeptidetransgene, e.g., encoding complement receptor 1 (CR1) using a lentivirusas described herein. Cells are cultured to terminal differentiation asdescribed herein. The presence of the exogenous protein in the cells isassessed by flow cytometry as described herein. The cells are labeledwith a fluorescent die, e.g., CFSE (Sigma Aldrich) per manufacturer'sinstructions to aid in their detection. The cells are injected into aNZBWF1/J mouse model of lupus, or other appropriate model of disease oractivity corresponding to the suitable receiver polypeptide,approximately 1×10̂8 cells injected via the tail vein. Blood is collectedat multiple time points by submandibular puncture. Immune complex levelsin the plasma are detected by Raji cell assay, see e.g., Theofilopouloset al. 976, J Clin Invest 57(1):169. Pharmacokinetics of the culturedcells are assessed by flow cytometry as described herein, by trackingthe percentage of CFSE fluorescent cells in the drawn blood sample.Mouse overall health is assessed by gross necropsy, including histologyof kidney tissue to track reduction of immune complex deposition andinflammation-mediated damage.

Example 21: Rapid Screening

Cell lines, e.g., 293T and K562, have shorter expression and culturingcycles (˜1 day) compared to cultured erythroid cells (days-weeks). Thesecell lines can be used to rapidly iterate through a gene libraryencoding suitable receiver polypeptides to identify the receiverpolypeptide with the highest expression or activity.

A library of suitable receiver polypeptide transgenes, e.g., full-lengthand shorter variants of complement receptor 1 (CR1), are constructed bypolymerase chain reaction and Gibson assembly as described herein. Thelibrary of transgenes is transfected into HEK293T cells in a parallelfashion in a microtiter plate using lipofectamine as described hereinand transduced into K562 cells using lentivirus as described herein. Theexpression of the receivers is assessed by flow cytometry as describedherein after 24-48 hours. The activity of each of the receivers in thelibrary is assessed by capture of fluorescent immune complex detectedwith flow cytometry as described herein, and by the transfer offluorescent immune complexes to cultured monocytes detected with flowcytometry as described herein. The receivers from the library that aremost functional—e.g., are highest expressed, capture most immunecomplexes, or best transfer immune complexes to monocytes—are thenindividually transduced into parallel erythroid cell cultures asdescribed herein using lentivirus as described herein. The expression ofeach receiver on cultured erythroid cells is assessed by flow cytometryas described herein The activity of each receiver on cultured erythroidcells is assessed by capture of fluorescent immune complex detected withflow cytometry as described herein, and by the transfer of fluorescentimmune complexes to cultured monocytes detected with flow cytometry asdescribed herein.

Example 22: Assessment of Clearance Rate of RBC In Vivo

The clearance rate of erythroid cells was assessed in vivo in animmunocompromised mouse model. NSG mice were treated at day −1 with 100uL of clordonate liposome (Clodrosomes.com) solution to selectivelydeplete macrophages. Cells were labeled with the fluorescent tag CFSEand approximately 1×10̂8 cells were injected into each mouse via the tailvein. At regular intervals blood was collected by submandibular punctureand blood cells were collected. Cells were co-stained with anti-humanGPA antibodies and analyzed by flow cytometry. Human erythroid cellswere distinguished from mouse erythroid cells by CFSE signal and byhuman GPA signal.

For therapeutic applications, it is important that cultured erythroidcells and cultured erythroid cells containing exogenous protein eitherintracellularly or on the surface circulate normally in vivo. This isshown in FIG. 9A-FIG. 9B using a standard immunocompromised mouse model.In FIG. 9A, blood collected from an injected mouse is analyzed on theflow cytometer. Cultured human erythroid cells are identified in the topright quadrant of the plot, double-positive for CFSE and human-GPA. InFIG. 9B, mice were injected with human red blood cells (solid circles),cultured enucleated erythroid cells (dashed diamonds), culturedenucleated erythroid cells that express an intracellular exogenousprotein (dotted squares) and cultured enucleated erythroid cells thatexpress a surface exogenous protein (open triangles). The clearance rateof the human cells is measured as the percentage of CFSE+ cellsremaining over time, scaled to the initial time point (20 minutes postinjection). There is no significant difference in clearance rate betweenthe four samples.

These data clearly demonstrates that cultured enucleated erythroid cellshave substantially similar circulation to normal human red blood cells.Furthermore, exogenous proteins expressed either in the intracellularspace or on the surface of the cells do not substantially affect thecirculation behavior of these cells. This is an important result fortherapeutic translation of the technology.

Example 23: Assessment of Adverse Circulatory Events

The incidence of adverse events caused by cultured eyrthroid cells incirculation were assessed by detection of fibrinogen breakdown productsin blood and histology in animals injected with cultured erythroidcells.

Detection of Fibrinogen Breakdown Products. Mice were injected withcultured erythroid cells as described herein. Blood was collected frommice by submandibular puncture in an EDTA-containing tube. Cells wereseparated by centrifugation and plasma was collected. The levels offibrinogen breakdown products fibrinopeptide A and fibrinopeptide B weremeasured in mouse plasma by ELISA (MyBiosource) following manufacturer'sinstructions.

Histology. Tissue samples from the same mice were collected followingnecropsy. Tissues were trimmed, embedded in paraffin wax, and sectioned.Tissue sections were stained by H&E staining and trichrome staining.Microscope images were taken at 10× and 20× magnification.

For therapeutic applications, it is important that cultured erythroidcells and cultured erythroid cells that contain exogenous proteins(either intracellularly or on the surface) not induce adverse events,such as activation of the clotting cascade and tissue thrombusformation.

FIGS. 10A and 10B show the levels of fibrinopeptide A and B in mouseplasma for mice injected with (1) human red blood cells, (2) culturedenucleated erythroid cells, (3) cultured enucleated erythroid cellsexpressing an intracellular exogenous protein, (4) cultured enucleatederythroid cells expressing a surface exogenous protein, and (5)recombinant protein alone. The levels of fibrinopeptide A and B, amarker of fibrinogen breakdown and activation of the clotting cascade,are substantially similar for all samples.

FIG. 10C and FIG. 10D show histologically stained sections of spleen fora mouse injected with cultured enucleated erythroid cells (FIG. 10C) andrecombinant protein (FIG. 10D). There is no substantial differencebetween the tissue, and no identifiable tissue damage in spleen, liver,lung, brain, heart, and kidney was observed between any of the samples.

These data conclusively demonstrate that cultured erythroid cells, withor without exogenous protein, do not induce any adverse events while incirculation in mice.

Example 24: Assessment of Exogenous Protein Retention in Circulation

The retention of exogenous proteins in and on cultured enucleatederythroid cells was assessed by flow cytometry and Western blotting.

1. Retention of Exogenous Protein Assessed by Flow Cytometry

Erythroid cells were cultured as described herein. A transgene constructencoding glycophorin A signal sequence, antibody scFv specific tohepatitis B surface antigen, HA epitope tag, and glycophorin A codingsequence was assembled by Gibson assembly as described herein. Thetransgene was introduced into the erythroid cells by lentiviraltransduction as described herein. The cells were cultured to terminaldifferentiation as described herein. Cells were fluorescently labeledwith CFSE and injected into an immunocompromised NSG mouse (JacksonLabs) via the tail vein (1×10̂8 cells per mouse). At regular intervalsblood was collected by submandibular puncture. Collected cells werestained with a fluorescent anti-HA antibody (Abcam), and analyzed byflow cytometry. Human cells were identified as CFSE+ cells, andexogenous protein retention was assessed by the fraction of CFSE+ cellsthat also stained positive for the epitope tag.

2. Retention of Exogenous Protein Assessed by Western Blot

Erythroid cells were cultured as described herein. A transgene constructencoding adenosine deaminase and an HA epitope tag was assembled byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Thecells were cultured to terminal differentiation as described herein.Cells were fluorescently labeled with CFSE and injected into animmunocompromised NSG mouse (Jackson Labs) via the tail vein (1×10̂8cells per mouse). At regular intervals blood was collected bysubmandibular puncture. Collected cells were washed, lysed, and analyzedby Western blot as described herein with a detection antibody againstthe HA epitope tag.

For therapeutic applications, it is important that cultured erythroidcells that contain exogenous proteins either intracellularly or on thesurface retain these transgenes when in circulation. This feat isnon-trivial given that it is widely hypothesized in the field thaterythroid cells undergo a rigorous program of maturation and eliminationof proteins unnecessary for basic function when in circulation as theymature (Liu J et al. (2010) Blood 115(10):2021-2027, Lodish H F et al.(1975) Developmental Biology 47(1):59).

FIG. 11A-FIG. 11B shows that exogenous proteins expressed in and oncultured enucleated erythroid cells were retained in circulation. InFIG. 11A, mice were injected with cultured enucleated erythroid cellsthat expressed antibody scFv on their surface. The percentage ofantibody scFv-positive cells began and remained steadily atapproximately 50% through the duration of the multi-day circulationstudy. In FIG. 11B, mice were injected either with cultured enucleatederythroid cells that expressed a cytoplasmic enzyme with an HA tag orwith recombinant enzyme with an HA tag. When analyzed by Western blot,it is clear that the enzyme retained within the cultured cell for theduration of the experiment. The decrease in band intensity isattributable to the clearance of cells during the experiment, not fromthe removal of exogenous enzyme from said cells.

The data clearly demonstrate that exogenous proteins expressed in and onculture enucleated erythroid cells are retained in and on the cells incirculation, which has tremendous and unprecedented implications fortherapeutic relevance.

Example 25: Assessment of Half-Life Extension In Vivo

Erythroid cells were cultured as described herein. A transgene constructencoding adenosine deaminase and an HA epitope tag was assembled byGibson assembly as described herein. The transgene was introduced intothe erythroid cells by lentiviral transduction as described herein. Thecells were cultured to terminal differentiation as described herein.Cells were fluorescently labeled with CFSE and injected into animmunocompromised NSG mouse (Jackson Labs) via the tail vein (1×10̂8cells per mouse). At regular intervals blood was collected bysubmandibular puncture. Collected cells were washed, lysed, and analyzedby Western blot as described herein with a detection antibody againstthe HA epitope tag.

A transgene encoding adenosine deaminase with an HA tag at theC-terminus was constructed by Gibson assembly as described herein. Thetransgene was introduced into HEK-293T cells by lipofectaminetransfection (Life Technologies) as described herein. The protein waspurified from the cell culture supernatant after 7 days using an HAaffinity resin (Pierce) according to manufacturer's instructions.Protein concentration was assessed by absorbance of light at 280 nm.Protein (40 ug) was injected into an immunocompromised NSG mouse(Jackson Labs) via the tail vein. At regular intervals blood wascollected by submandibular puncture. Plasma was analyzed by Western blotas described herein with a detection antibody against the HA epitopetag.

In FIG. 11B, mice were injected either with cultured enucleatederythroid cells that expressed a cytoplasmic enzyme with an HA tag orwith recombinant enzyme with an HA tag. When analyzed by Western blot,it is clear that the enzyme's circulating half-life is significantlyextended when expressed within a circulating cell compared to wheninjected in soluble form.

Example 26: Assessment of Clearance Rate In Vivo—Platelets

A population of exogenous thymidine phosphorylase expressing plateletsis cultured using the herein detailed procedure and is labeled with CFSEand injected into an NSG mouse via the tail vein. A population of nativehuman-sourced platelets is similarly labeled with CFSE and injected intoanother mouse. Samples are taken from both mice at 10 min, 1 h, 4 h, 8h, 24 h, and 48 h and flow cytometry is used to quantify plateletcirculation levels. The half-life of natural vs cultured platelets iscompared.

Example 27: Assessment of Adverse Circulatory Events—Platelets

For therapeutic applications, it is important that cultured plateletsand cultured platelets that contain exogenous proteins (eitherintracellularly or on the surface) not induce adverse events, such asactivation of the clotting cascade and tissue thrombus formation. Uponinjection of cultured platelets into an NSG mouse via the tail vein,fibrinogen breakdown products fibrinopeptide A and fibrinopeptide B aredetected in mouse plasma by ELISA following manufacturer's protocol(MyBiosource). Tissue samples from NSG mice are collected followingnecropsy. Tissues are trimmed, embedded in paraffin wax, and sectioned.Tissue sections are stained by H&E staining and trichrome staining.Microscope images are taken at 10× and 20× magnification and assessed bya trained pathologist for any pathogenic features.

Example 28: Assessment of Exogenous Protein Retention inCirculation—Platelets

The retention of exogenous proteins in and on cultured platelets isassessed by flow cytometry and Western blotting.

CFSE labeled platelets that contain intracellular exogenous protein areinjected into a mouse via the tail vein. At regular intervals blood iscollected by submandibular puncture. Blood is centrifuged to isolate theplatelet-rich plasma, which is then lysed, and analyzed by Western blotwith staining for an epitope tag present on the exogenous protein.

Example 29: Acquisition of Donor Cells for Production

After obtaining informed consent, healthy CD34+ stem cell donors receiverhG-CSF (Granocyte or Neupogen), 10 ug/kg/day s.c., for 5 days forperipheral blood stem cell mobilization and then undergo apheresis for 2consecutive days to collect mobilized CD34+ HSC. Mononuclear cells (MNC)are isolated from mobilized peripheral blood by Ficoll density gradientcentrifugation and are split in two parts. One part is used to purifyCD34+ cells by using anti-CD34-coated magnetic beads (Miltenyi Biotec,Inc., Germany), relative to Miltenyi protocol. The purity of the CD34+fractions is controlled. CD34+-enriched HSC are then used immediately inthe two-step culture method or frozen until use in the one-step culturemethod.

Complete medium (CM) used is RPMI 1640 (Eurobio, France), supplementedwith 2 mM L-glutamine and 100 IU/ml penicillin-streptomycin (Gibco,Grand Island, N.Y., USA) and 10% heat-inactivated FBS (Gibco). IMDM(Gibco), supplemented with 10% heat-inactivated FBS, is used forexpansion. Recombinant human stem cell factor (rhSCF), thrombopoietin(TPO), fetal liver tyrosine kinase 3 ligand (Flt-3L), GM-CSF, andTNF-alpha are purchased from R&D Systems (Minneapolis, Minn., USA).

Example 30: Scale-Up for Production

Erythroid cells are scaled up in volume progressively, maintaining thecells at a density of between 1×10̂5 and 2×10̂6 cells/mL in staticculture. Expansion stage is seeded at 10̂5/ml and includes 3-7progressive volume transfers; 100 ml, 500 ml, 1 L, 10 L, 50 L, 100 L,100 L. During the course of production the cell media includes acombination of IMDM, FBS, BSA, holotransferrin, insulin, glutamine,dexamethasone, beta estradiol, IL-3, SCF, and erythropoietin. When thecells reach a volume appropriate for seeding the production bioreactor,they are transferred to the production bioreactor for final scale-up anddifferentiation.

Example 31: Culturing Cells in a Bioreactor (Wave)

The WAVE Bioreactor 2/10 system is set up according to the operatormanual. In brief, the Cellbag is assembled on the rocking unit, which isplaced on the perfusion module. After inflating the bag with air, theweight is set to zero. Subsequently, the bag is filled with theappropriate amount of culturing media and incubated for at least twohours, allowing the media to reach 37 C. The media and cells aretransferred to the bag via a transfer flask, a special designed DURANglass bottle with two ports. In the upper part of the flask, a filter isconnected to the port. In the other port, by the bottom of the flask, atube is assembled. The tube one the transfer flask is coupled with thefeed connection on the Cellbag. The transfer flask is maintained in aLAF hood, to decrease the risk of contamination.

Before perfusion is started, tubing and containers for harvest and feedare connected to the Cellbag. Tubing is prepared as follows; a 50 or 70cm long Saniflex ASTP-ELP silicone tubing (Gore/Saniflex AB), with aninner and outer diameter of 3.2 respectively 6.4 mm, is equipped withmale luer lock connections in both ends. The silicone tubing isconnected to one end of a C-Flex tube, via a female luer lock. At theother end of the C-Flex tube a male luer lock is assembled and tubingsare thereafter autoclaved. Luer locks are held in place with zip-ties onall tubes. Prior to perfusion, the silicone part is connected to theCellbag and the C-Flex part to a 5 L container (Hyclone Labtainer) forboth feed and harvest. All connections are performed in a laminarairflow cabinet.

Control of environmental and metabolic factors can alter the expressionor activity of transcription factors and gene regulatory proteins oferythroid cells in culture, see e.g., Csaszar et al., 2009 BiotechnolBioeng 103(2):402; Csaszar et al. 2012 Cell Stem Cell 10(2):218. Toprovide control over inputs and outputs in the reactor a micro-volumedelivery system is created, a key component of which is a 60-80 cm longfused silica capillary (#TSP100375, Polymicro Technologies) with aninternal diameter of 100 um. At the input end, the capillary is fed witha luer-lok tip stock syringe (#309585 BD) connected via a PEEK luer to aMicroTight adapter (#P-662, Upchurch Scientific). The stock syringe isloaded on a Model 33 Twin Syringe Pump (#553333, Harvard Apparatus),kept in a refrigerator at 4 C. At the output end, the capillary entersthe bioreactor: a two port FEP cell culture bag (#2PF-0002, VueLife)placed on an orbital shaker in a cell culture incubator at 37 C with 5%CO2. The capillary is fed through a self-sealing rubber septa (#B-IIS,InterLink) with a needle, into the midpoint of the bioreactor. Theopposing connector on the bioreactor is replaced with an additionalself-sealing rubber septa. Stock syringes and delivery capillaries areblocked overnight before use with a solution of PBS with 10% fetalbovine serum to prevent protein adhesion to syringe and capillary walls.

National Instruments LabVIEW 7.1 is used to create a program to controlthe syringe pump's injections. The program's basic dosing strategy is aninitial injection to concentration L1 followed by wait time t1 andsubsequent injections, each to concentration L2 and followed by waittime t2, repeated for n times. The user inputs the flow rate, the stockconcentration, the initial culture volume, the desired concentrationafter injections, the time between injections, and the total number ofinjections.

Example 32: Assess Expansion and Differentiation of Cultured ErythroidCells

It is important to assess the expansion, differentiation, andenucleation in vitro differentiated cells to ensure that theintroduction of a transgene does not negatively affect the quality ofthe cells in culture. Expansion is assessed by cell counting.Differentiation is assessed by flow cytometry, Western blot, and RT-PCR.Enucleation is assessed by flow cytometry.

Assessing Expansion Rate by Cell Counting. Erythroid cells are culturedas described herein. At various time points, cells are collected, washedwith PBS, and counted using a Countess Automatic Cell Counter instrument(Life Technologies). The expansion rate of the cells is determined bythe growth in number of cells over time.

Assessing Differentiation by Flow Cytometry. Erythroid cells arecultured as described herein. At various time points, cells arecollected, washed with PBS, and stained with 1:100 dilutions offluorescent antibodies against the cell surface markers GPA (CD235a),CKIT (CD117), and TR (CD71), purchased from Life Technologies. Labeledcells were analyzed by flow cytometry as described herein.

Assessing Differentiation by Western Blot. Erythroid cells are culturedas described herein. At various time points, cells are collected, washedwith PBS, lysed with RIPA buffer, and analyzed by Western Blot asdescribed herein using antibodies for differentiation markers GATA1,GATA2, Band3, CD44, and actin (Abcam).

Assessing Enucleation by Flow Cytometry. Erythroid cells are cultured asdescribed herein. At various time points, cells are collected, washedwith PBS, and stained with a fluorescent antibody against glycophorin A(Life Technologies) and the nucleic acid stain DRAQ5 (Pierce) atmanufacturer-recommended dilutions, and analyzed on an Attune flowcytometer as described herein.

Assessing Enucleation by Microscopy (Benzidine-Giemsa). Erythroid cellswere cultured as described herein. At various time points, cells werecollected, washed with PBS, and spun onto slides using a Cytospin(Thermo Scientific). Cells were fixed cells after cytospin with −20 Cmethanol for 2 min at room temp, rinsed with water, and air-dried. Abenzidine tablet (Sigma#D5905) was dissolved with 10 mL PBS, to which 10μL of H2O2 was added. The solution was filtered with a 0.22 um syringefilter. The cell spot on the slide was covered with 300-500 uL ofbenzidine solution, incubated at room temperature for 1 hr, then washedwith water. Giemsa stain was diluted (Sigma#GS500) 1:20 with water. Thecell spot on the slide was covered with 300-500 uL Giemsa solution,incubated at room temperature for 40 minutes, washed with water, andair-dried. Slides were then mounted and sealed before imaging on amicroscope.

FIG. 12A shows the expansion rate of erythroid cells in culture during aseven day window of expansion and differentiation for cells that containtransgenes (dashed line and dotted line) and cells that do not contain atransgene (solid line). Of note, the expansion rate of cultured cellsthat contain a transgene is indistinguishable from that of cells that donot contain a transgene.

FIG. 12B is a collection of flow cytometry plots for cells stained withantibodies against the cell surface differentiation markers GPA andCKIT. At this particular stage of differentiation, the culture is losingits CKIT expression and increasing its GPA expression as the cellsapproach terminal maturation. Of note, cultured cells that contain atransgene are indistinguishable from those that do not contain atransgene by this metric of differentiation.

FIG. 12C is a collection of flow cytometry plots for cells stained withan antibody against the surface marker GPA and a fluorescent DNA stain.Three cell populations are evident: (1) cells that are GPA-high andDNA-low, comprising enucleated erythroid cells; (2) cells that areGPA-high and DNA-high, comprising erythroid cells that still containgenetic material; and (3) cells that are GPA-low and DNA-high,comprising pyrenocytes or the membrane-encapsulated ejected nuclei fromenucleated cells. Of note, cultured cells that contain a transgene areindistinguishable from those that do not contain a transgene by thismetric of enucleation.

The introduction of a transgene into cell culture does not noticeablyaffect the rate of expansion, the differentiation, or the rate ofenucleation of the cells in culture.

Example 33: Assess Hemoglobin Content

1. Total Hemoglobin

Erythrocyte hemoglobin content was determined by Drabkin's reagent(Sigma-Aldrich, product D5941) per manufacturer's instructions. Briefly,blood cells were combined with the reagent in an aqueous buffer, mixedthoroughly, and absorbance of light at a wavelength of 540 nm wasmeasured using a standard spectrophotometer. A soluble hemoglobinstandard curve was used to quantify the hemoglobin content in the cells.

2. Hemoglobin Typing by RT-PCR

Cells were lysed and total RNA is collected. Reverse Transcription wascarried out with the SuperScript First-Strand Synthesis System forRT-PCR (Life Technologies) according to manufacturer's protocol.Briefly, total RNA (5 ug) was incubated with 150 ng random hexamerprimer and 10 nmol dNTP mix in 10 uL H₂O for five minutes at 65 C then 1minute on ice. The reaction master mixture was prepared with 2 uL 10×RTbuffer, 4 uL of 25 mM MgCl2, 2 uL of 0.1 M DTT, and 1 uL of RNAseOUT.The reaction mixture was added to the RNA/primer mixture, mixed briefly,and then placed at room temperature for 2 min 1 uL (50 units) ofSuperScript II RT was added to each tube, mixed, and incubated at 25° C.for 10 min. The reaction was incubated at 42 C for 50 min, heatinactivated at 70 C for 15 min, then stored on ice. 1 uL RNase H wasadded and incubated at 37 C for 20 min. This reaction product, the1^(st) strand cDNA, was then stored at −20 C until needed for RT-PCRreaction.

Primers to amplify the different hemoglobin genes and control genes werepurchased from IDT-DNA. The primers were as follows:hHBB_F—tcctgaggagaagtctgccgt (Seq. ID No. 9);hHBB_R—ggagtggacagatccccaaag (Seq. ID No. 10);hHBA_F1—tctcctgccgacaagaccaa (Seq. ID No. 11);hHBA_R1—gcagtggcttagcttgaagttg (Seq. ID No. 12);hHBA_F2—caacttcaagctaagccactgc (Seq. ID No. 13);hHBA_R2—cggtgctcacagaagccag (Seq. ID No. 14);hHBD_F—gactgctgtcaatgccctgt (Seq. ID No. 15);hHBD_R—aaaggcacctagcaccttctt (Seq. ID No. 16);hHBG2_F—cactggagctacagacaagaaggtg (Seq. ID No. 17);hHBG2_R—tctcccaccatagaagataccagg (Seq. ID No. 18);hHBE_F—aagagcctcaggatccagcac (Seq. ID No. 19);hHBE_R—tcagcagtgatggatggacac (Seq. ID No. 20);h18S-RNA-F—cgcagctaggaataatggaatagg (Seq. ID No. 21);h18S-RNA-R—catggcctcagttccgaaa (Seq. ID No. 22).

An RT PCR reaction mix was prepared with 25 uL SYBR Green Mix (2×)(Applied Biosystems), 0.5 uL 1^(st) strand cDNA, 2 uL forward/reverseprimer pair mix (each primer at 5 pmol/uL), in a total volume of 50 uLH2O. Reactions were run in an ABI Prism SDS 7000 instrument (appliedbiosystems) using the following amplification cycle: 50 C 2 min, 1cycle; 95 C 10 min, 1 cycle; 95 C 15 s->60 C 30 s->72 C 30 s, 40 cycles;72 C 10 min, 1 cycle. Dissociation curve analysis and RT-PCR results wasperformed with the SDS 7000 instrument.

Example 34: Assess Differentiation of Cultured Platelets—FACS

The differentiation state of platelets in culture can be assessed byflow cytometry. Megakaryocytes (MKs) represent a distinct cellularmorphology that precedes terminal platelet differentiation. To determinethe extent of maturation toward MKs, 1×10̂6 cultured cells (LAMA-84 andCD34+ cells) are washed and then labeled with (a) anti-CD41-FITC(GpIIb/IIIa; BD Bioscience, San Jose, Calif., USA) or anti CD71-FITC or(b) anti-CD33-FITC, anti-CD41-PE, anti-CD45-PerCp and CD34-APC (BeckmanCoulter, Fullerton, Calif., USA), and analyzed for the percentage ofCD41 cells generated.

To determine the amount of ploidy, differentiated LAMA-84 cells arefixed overnight in 75% ethanol at 4° C. and labeled with propidiumiodide (PI, 50 μg/ml) and analyzed using the FACScalibur (BectonDickinson), whereas day 14 differentiated CD34+ cells are analyzedquantitatively under a microscope after May-Grunwald/Giemsa staining byquantitating the number of nuclei per cell and specific morphology ofMKs with this stain. Only cells with MK morphology are analyzed. Thepresence of multinucleated cells in the cytospin preparation isindicative of the presence of polyploid MKs. Differentiated CD34+ cellsare assessed for the presence of multinucleated mature MKs bymorphology.

Example 35: Assess Differentiation of Cultured Platelets—qPCR

The differentiation state of platelets in culture can be assessed byquantitative PCR. Platelet RNA is extracted to further characterize thecultured cells. Total RNA is extracted using TRIzol reagent(Invitrogen). The purity of each platelet preparation is assessed by PCRanalysis of platelet (GPIIIa) and leukocyte (CD45) markers. Theintegrity of platelet RNA is assessed using Bioanalyzer 2100 (Agilent)prior to further analyses.

Total RNA is collected from cell lysate and a cDNA library is generatedusing a commercial synthesis kit (Clontech). The labeled cDNAs arequantified with the Quant-iT PicoGreen dsDNA Kit (Invitrogen) anddiluted to 3 pM for loading into a single lane and sequencing on anIllumina 1G Genome Analyzer (Solexa).

Raw sequences are filtered through serial quality control criteria.First, the presence of at least 6 nt of the 3′ Solexa adapter isverified. The sequence reads that did not comply with this criterion arediscarded, whereas the others are trimmed to remove the adapter sequenceharbored at the 3′ end. The remaining tags are further filteredregarding their length (>10 nt), copy number (>4 reads) and readability(<9 non-identified nucleotides, annotated N). Reads complying with allthose criteria are subsequently defined as usable reads.

All the usable reads are aligned to pre-microRNAs extracted from miRBasedatabase. Sequence tags that matched perfectly to more than oneprecursor are distributed equally among them. In order to account forDrosha and Dicer imperfect cleavage, any sequence tag that perfectlymatched the pre-microRNA in the mature microRNA region, allowing up to 4nt shift as compared to the reference mature microRNA position, isconsidered as a mature microRNA. The microRNA expression level isdefined as the number of reads mapping each mature microRNA normalizedto the total number of usable reads, considering that the overall numberof small RNAs is invariant. The relative abundance of each microRNA isdefined as the number of reads mapping each microRNA compared to thetotal number of reads mapping mature microRNAs.

Example 36: Purification by Centrifugation

Cultured cell fractions can be purified and separated from nuclei andcontaminating alternate-density cell types via centrifugation. Cells arecentrifuged at 200 g for 15 minutes to isolate an erythrocyte andreticulocyte rich fraction. The supernatant is pipetted off and thedesirable cell fraction is then washed in modified Tyrode's buffer(containing 138 mM NaCl, 5.5 mM dextrose, 12 mM NaHCO3, 0.8 mM CaCl2,0.4 mM MgCl2, 2.9 mM KCl2, 0.36 mM Na2HPO4 and 20 mM Hepes, pH 7.4) inpresence of 1 μM prostaglandin I2, and resuspended in the same buffer.

Example 37: Purification by Chemical Enucleation

Enucleation of cultured cells can be stimulated by chemical additives tothe culture, which can help increase the enucleated fraction of cellsprior to purification. Erythroid cells are cultured as described herein.48 hours prior to collection, cells are incubated with 210 mM Me2SO.Cells are then collected by centrifugation at 350×g for 5 min at roomtemperature, resuspended at a level of 3×105 cells per ml in freshmedium containing 210 mM Me2SO and 5 ug/mL of cytochalasin B (or otheractin or nucleus manipulating molecule, ie. p38 MAPK, psoralens) andincubated at 37 C. The proportion of cells without nuclei is assessed byflow cytometry as described herein, using DRAQ5 as a nucleic acid stainand antibodies against glycophorin A as an erythroid surface marker ofdifferentiation.

Example 38: Purification by Acoustophoresis

Several mechanical separation systems may be used to obtain a uniformcell population. Free flow acoustophoresis represents one mechanicalseparation method (Petersson 2007, American Chemical Society). Whilesuspended in saline solution (0.9 mg/mL) with nutrient additives,including CsCl (0.22 g/mL), is added to the saline solution. A samplesuspension containing cultured erythroid cells is processed using anacoustopheresis chip (Cell-Care) with two active outlets (flow rate 0.10mL/min per outlet).

Syringe pumps (WPI SP260P, World Precision Instruments Inc., Sarasota,Fla.) are used to control the flow rates in the chip. All outlets areindividually connected to high-precision glass syringes (1005 TLL and1010 TLL, Hamilton Bonaduz AG, Bonaduz, Switzerland) via the injectorsusing Teflon tubing, allowing independent control of the outlet flowrates. The clean fluid inlet is connected to a syringe pump and the cellsuspension inlet to a 50-mm-long piece of Teflon tubing (0.3-mm i.d.)with its other end submerged in a beaker from which the samplesuspension is aspirated at a rate defined by the difference between thenet outlet flow and the clean fluid inlet flow.

The ultrasound used to induce the standing wave between the walls of theseparation channel is generated using a 20×20 mm piezoelectric ceramic(Pz26, Ferroperm Piezoceramics AS, Kvistgard, Denmark) attached to theback side of the chip. Ultrasonic gel (Aquasonic Clear, ParkerLaboratories Inc., Fairfield, N.J.) ensures a good acoustic couplingbetween the two. The piezoelectric ceramic is actuated via a poweramplifier (model 75A250, Amplifier Research, Souderton, Pa.) connectedto a function generator (HP 3325A, Hewlett-Packard Inc., Palo Alto,Calif.). Even though the acoustic waves enter the chip from the backside, a standing wave is induced between the side walls of theseparation channel as a result of the coupling of the mechanicalvibrations along the three axes of the crystal structure.

The separation process is monitored using a standard microscope and awattmeter (43 Thruline Wattmeter, Bird Electronic Corp., Cleveland,Ohio). The process can subsequently be controlled by tuning the signalfrequency, the actuation power, and the flow rates.

The cell size distributions in the samples are analyzed using a Coultercounter (Multisizer 3, Beckman Coulter Inc., Fullerton, Calif.). Eachsample is mixed with an electrolyte (Isoton II, Beckman Coulter Inc.)and analyzed using a 100-um aperture. The level of hemolysis, i.e., theconcentration of free hemoglobin from damaged red cells, is measuredusing a photometer (Plasma/low HB Photometer, HemoCue AB, Angelholm,Sweden).

Example 39: Purification by Ex Vivo Maturation

Erythroid cells that are not fully mature can be driven to maturity byex vivo incubation in a system that mimics the natural in vivomaturation triggers.

1. Co-Culture with Stromal Cells

In the final stage of culture, erythroid cells are cultured on anadherent stromal layer in fresh medium without cytokines. The culturesare maintained at 37 C in 5% CO2 in air. The adherent cell layerconsists of either the MS-5 stromal cell line or mesenchymal stromalcells (MSCs) established from whole normal adult bone marrow (seeProckop, DJ (1997) Science 276:71) in RPMI (Invitrogen) supplementedwith 10% fetal calf serum. Adherent MSCs are expanded and purifiedthrough at least two successive passages prior to use in co-culture.

2. Culture in Fibronectin-Coated Plates

In the final stage of culture, erythroid cells are cultured in platesadsorbed with human fibronectin. To produce these plates, fibronectin(Sigma Aldrich) is reconstituted with 1 mL sterile H2O/mg of protein andallowed to dissolve for at least 30 minutes at 37° C. A small amount ofundissolved material may remain. This will not affect productperformance. The fibronectin solution is diluted 100× in sterilebalanced salt solution and added to the culture surface with a minimalvolume. The culture surface is allowed to air dry for at least 45minutes at room temperature. Excess fibronectin is removed byaspiration.

Example 40: Purification by Magnetophoresis

Strategies for separating, enriching, and/or purifying erythroid cellsby magnetophoresis are known in the art, see e.g., Zborowski et al.,2003, Biophys J 84(4) 2638 and Jin & Chalmers 2012, PLOS One 20127(8):e39491. A commercial magnetic separation system (QuadroMACS™Separator combining four MidiMACS™ separation units and LD columns,Miltenyi Biotec, Auburn, Calif.) is used for magnetic erythrocyteenrichment from HSC-derived erythrocyte cultures. Cells are deoxygenatedin a Glove-Bag™ inflatable glove chamber (Cole Parmer, Vernon Hills,Ill.), filled with nitrogen (Medipure™ nitrogen, concentration >99%,Praxair, Inc., Danbury, Conn.). Before deoxygenation, all materials andequipment including the separation system, degassed sterile buffer(PBS+2 mM EDTA+0.5% BSA), and sterile collection tubes are placed in theglove bag, which is then tightly sealed. Deoxygenated cultures areloaded directly into a MACS® LD column which was placed in theQuadroMACS™ separator kept under anoxic conditions inside an inflatableglove chamber filled with N2 gas. Cells which pass through the columncontained within the magnet are labeled as negative fraction and theyare expected to be “non-magnetic”, including HSCs and erythroid cellsbefore final maturation. The cells retained in the separation column arelabeled as positive fraction, which is “magnetic” and consist ofmaturing RBC-like cells nearly full of functional hemoglobin. They areeluted from LD column after its removal from the magnet. Once separationis finished, oxygenated cells are reversibly recovered by exposing thecollected cells to air.

Example 41: Purification by FACS

A population of erythroid cultured cells is sorted using aBecton-Dickinson Aria IIu cell sorter. Prior to sorting, cells arecollected, washed with PBS, and stained with a fluorescent antibodyagainst glycophorin A (Life Technologies) and the nucleic acid stainDRAQ5 (Pierce) at manufacturer-recommended dilutions. A 100 μm nozzle isused with a drop drive frequency of 28,000 drops/second. The samplethreshold rate is approximately 4000 events/second. The temperaturecontrol option is used to maintain sample and collection tubes at 4° C.the entire duration of sorting. Additionally, the sample agitationfeature is used at 200 rpm to prevent the sample from sedimentingthroughout the sort. The sample is sorted in aliquots of approximately750 μl dispensed from the syringe. Meanwhile, during these pauses thecollection tubes are kept at 4° C., protected from the light, and gentlymixed prior to resuming sort. The sorted samples are collected into a12×75 mm borosilicate glass collection tube containing 250 μl DMEMsupplemented with 10% FCS.

Example 41: Purification by Enzymatic Treatment of Cells

Allogeneic erythrocyte sourcing may benefit from A and B antigen removalto generate a universally compatible product. This may be facilitated bya set of enzymes capable of selectively cleaving the galactose groups,rendering the erythroid cells more immunogenically favorable.

Two types of recombinant proteins of endo-β-galactosidase, which areoriginally identified from Clostridium perfringens, are produced in E.coli BL-21 using standard cloning methods. ABase is prepared forreleasing A/B Ag and endo-β-galactosidase C (EndoGalC) for releasingGalα1-3Galβ1-4GlcNAc (Gal Ag), which is known to be highly immunogenicin xenotransplantation, and has a carbohydrate structure resembling theA/B Ag. ABase cleaves Galβ1-4GlcNAc linkage in blood type A[GalNAcα1-3(Fucα1-2) Galβ1-4GlcNAc] and in blood type B[Galα1-3(Fucα1-2) Galβ1-4GlcNAc].

Briefly, after cloning of ABase, an expression plasmid with a C-terminalHis tag is constructed in the pET-15b vector eabC without signalpeptide. This exogenous gene is transformed into E. coli BL-21 cells.The enzyme produced in the cells as a soluble protein fraction ispurified over a nickel-nitrilotriacetic acid column (QIAGEN GmbH,Hilden, Germany). Finally, 5 mL of purified recombinant ABase isobtained at the concentration of 3.6 mg/mL with the specific activity of1500 U/mg. One unit of the enzymatic activity is defined as the amountof the enzyme required to hydrolyze 1 μmol of the substrate per min.

The effect of ABase treatment on Ag presence, Ab binding and complementactivation is examined Human A/B RBC are digested with ABase andsubjected to flow cytometric analysis after incubation withcross-reactive (anti-A or anti-B or anti-A and B containging; type B,type A or type O respectively) human sera. The mean fluorescenceintensity (MFI) is used to quantitate the expression level of blood typeA, B and Gal Ag. Digestion level is expressed as a percentage of bloodtype A or B Ag expressed on RBC after incubation in the absence ofABase.

Fresh blood type O sera are pooled from three healthy human volunteersand frozen at −80° C. to preserve endogenous complement activity untilused. Heat-inactivated (for 30 min at 56° C.) sera are used for analysisof Ab binding. RBC with and without enzyme (ABase) digestion areincubated with 50% blood type O sera (100 μL) diluted withphosphate-buffered saline containing 0.2% bovine serum albumin (PBS/BSA)for 30 min at 37° C. After washing, RBC are reacted with FITC-labeledanti-human IgG/IgM (DAKO, Glostrup, Denmark) (×30, 100 μL) for 30 min at4° C. and then subjected to flow cytometric analysis.

The inhibitory effect of enzyme treatment on complement activation isalso evaluated by the change of C3d deposition. After RBC are incubatedwith 50% human sera in the presence of complement activity for 15 min at37° C., RBC are reacted with FITC-labeled rabbit anti-human C3d Ab(DAKO, Glostrup, Denmark) (×100, 100 μL) for 30 min at 4° C. and thenapplied to flow cytometric analysis. The percentage of the control level(in the absence of enzyme) is calculated based on MFI to evaluate theinhibitory effect of enzyme treatment on Ab binding and C3d deposition.

Example 42: Purification of Platelets by Centrifugation

Platelets can be purified from mixed cell suspensions by centrifugation.Some 40 ml of whole blood is distributed in blood collection tubes withsodium citrate at 3.2% used as an anticoagulant. The tubes arecentrifuged at 400×g for 10 min. After this stage, three layers areclearly demarcated: plasma, red blood cells, and an intermediate zone.The plasma is at the top with the platelets, the red blood cells are atthe bottom because of their heavier density; and the fine, whitishintermediate zone consists of larger platelets and leukocytes and iscalled the buffy coat. Using a Jelco 18G needle, the upper portion ofplasma with platelets is drawn off, and the buffy coat is placed intotwo other tubes, this time with no additives: one tube to produce plasma(P tube) and the other to produce thrombin (T tube). Only 1.5 ml ofplasma is used to produce thrombin, to which 0.5 ml of calcium gluconateat 10% is added, with 15 min in a double boiler at 37° C. The two tubesare then centrifuged again, this time at 800×g, for the same length oftime (T=10 min). After this final centrifugation, the T tube contains athrombin-rich liquid while the P tube contains the plateletsedimentation and some red blood cells (erythrocyte-platelet clump). Thevolume is reduced at this stage by removing two-thirds of the totalplasma volume. The portion removed is platelet poor, while the remainingportion with the sedimented platelets (that are easily dispersible bystirring) is platelet rich.

Example 43: Thymidine Incorporation

Self-replication potential of a cell population can be assessed using athymidine incorporation assay known in the art, see e.g., Harkonen etal. 1991 Exp Cell Res 186L288 and Tanaka et al. 1992 PNAS 89:8928.

Briefly, uniformly 13C- and 15N-enriched thymidine [U-13C, 15N-TdR] isobtained from Martek Biosciences (Columbia, Md.), and 3 H-TdR (80Ci/mmol) is purchased from ICN Radiochemicals (Irvine, Calif.). Mediaand buffers are obtained from Fisher Scientific (Pittsburgh, Pa.). Allenzymes except phosphodiesterase are from Boehinger Mannheim(Indianapolis, Ind.). Phosphodiesterase II is obtained from WorthingtonBiochemical Corporation (Lakewood, N.J.). High-performance liquidchomatography (HPLC) solvents are from EM Science (Gibbstown, N.J.) andcontained <0.1 ppm evaporation residue.

Erythroid cells are cultured as described herein. Following the culture,cells are collected for use in the thymidine incorporation assay.

Cells are labeled with [U-13C, 15N]-TdR at 1.6 μg/ml for 18 h, with theaddition of unenriched thymidine to achieve a final thymidineconcentration of 1 μM. After they are washed with phosphate-bufferedsaline, the cells are cultured in supplemented DMEM for 6 h more before3 H-TdR is added at the indicated concentrations (0.1-10 μCi/ml) foranother 18-h incubation. Unlabeled thymidine is added to the samples tobring the final thymidine concentration to 0.13 μM, which is equivalentto the concentration of 3 H-TdR in the samples receiving 10 μCiradiolabel/ml. After removal of 3 H-TdR, the cells are incubated insupplemented DMEM for an additional 6-54 h before isolation of DNA.

DNA is extracted using the modified Puregene DNA isolation kit (GentraSystems, Minneapolis, Minn.). Based on the number of cells in thesample, a scale-up/scale-down procedure is used to determine the addedreagent volumes. For example, when 1×10̂7 cells are used, 21 μlcontaining 328 μg of proteinase K is added to 3 ml of cell lysissolution. After mixing, the sample is left overnight at roomtemperature. The following day, 10 μg of RNase is added and the sampleis mixed and incubated for 2 h at 37 C. Protein precipitation solution(1 ml) is added, and the sample is incubated on ice for 5 min Aftercentrifugation for 10 min at 2000 g, the supernatant containing DNA ismixed with 3 ml 100% 2-propanol and gently inverted 50 times or untilwhite threads of DNA became visible. The sample is then centrifuged at2000 g for 5 min. The resultant DNA pellet is dried for 5 min beforewashing in 3 ml of 70% ethanol and recentrifugation for 5 min at 2000 g.The final pellet is air-dried and then rehydrated in deionized H₂O andquantitated by absorption at 260 nm. The same procedure is applied toCD34+ stem cells as a control for replicative ability.

Any DNA is denatured by boiling for 3 min, then chilled rapidly on ice.The enzymatic hydrolysis procedure is carried out with a DNAconcentration of 0.5 mg/ml. The following protocol describes volume ofreagent added per milliliter of DNA solution. DNA is hydrolyzed with 10μl of nuclease P1 (0.5 U/μ1) and 5 μl of DNase I (4 U/μ1) in 10 μl ofbuffer containing 200 mM MgCl2, 100 mM ZnCl2, and 1 M Tris, pH 7.2, for2 h at 45° C., followed by addition of 20 μl phosphodiesterase (4 mU/μ1)and further incubation for 2 h at 37° C. Finally, 5 μl of 10 M ammoniumacetate (pH 9.0) and 10 μl of alkaline phosphatase (1 U/μ1) are added,and the samples incubated for another 2 h at 37° C.

The digested DNA sample is filtered with a 0.22-μm nylon filter. Thissample is analyzed with the HPLC/CRI/IRMS system, using a 4.6×250 mmSupelcosil LC-18-S HPLC column (Supelco, Bellefonte, Pa.). The samesolvent system is used at 1 ml/min and a linear gradient of 5% to 25% Bin 15 min.

After separation by HPLC, the deoxynucleosides are analyzed usingchemical reaction interface mass spectrometry (CRIMS). In this process,the deoxynucleosides flow into a nebulization and desolvation systemdriven by a stream of helium, where they emerge as a dry particle beam.The 13CO2/12CO2 abundances from this in-line generated CO2 aredetermined with a Finnigan/MAT Delta S isotope ratio mass spectrometer(ThermoFinnigan, San Jose, Calif.) and its accompanying Isodat datasystem. 5-Fluorodeoxyuridine (Sigma) is used as an internal isotoperatio standard.

Isotope ratios (IR in equation that follows) for three nucleosides areobtained from each sample: T, dA, and dG. The enrichment of CO2 evolvedfrom each DNA-derived deoxynucleoside is computed by the equation(13)CO2 (per mil)=1000×(IR experimental−IR std)/IRstd. To maintain thehighest level of internal consistency and avoid any interexperimentaldrift, the isotope ratio for dG is subtracted across all experimentsfrom the isotope ratio for T. The data from the end of thestable-isotope labeling period (day 0) to the end of the washout (day 3)are evaluated.

Example 44: Quantification of Nuclear Material

The number of cells in a mixed population that contain DNA is assessedby flow cytometry using the DNA stain DRAQ5 (Pierce). Cells areincubated with the stain per manufacturer's instructions and analyzed ona flow cytometer, e.g., an Attune cytometer (Life Technologies). Thepercentage of cells above a predefined threshold of nuclear materialcontent is quantified.

Example 45: Tumorigenicity Assay In Vitro

To assess the replication potential of cells, a soft agar colonyformation assay can be performed. In brief, a base agar layer is made bymaking a 0.5% Agar+1×RPMI+10% FCS solution, all components warmed to 40C, and adding 1.5 mL of the solution to a 35 mm petri dish. The agar isallowed to solidify for 30 min at room temp before use.

The top agarose layer is prepared by melting 0.7% agarose in a microwaveand cooling to 40 C. A 2×RPMI+20% FCS solution is heated to 40 C. Cellsare counted and prepared for plating at 5000 cells per plate at adensity of 200,000 cells per mL. 0.1 mL of cell suspension is added to10 mL tubes, followed by 3 mL of the warm 0.7% Agarose and 3 mL of thewarm RPMI/FCS solution. The solution is mixed gently by swirling andadded (1.5 mL) to each of three or four replicate base agar plates.

Plates are incubated at 37 C in a humidified incubator for 10-30 days.Cells are fed 1-2 times per week with cell culture media, 0.75 mL/plate.

To assess colony formation, plates are stained with 0.5 mL of 0.005%Crystal Violet for >1 hr. Colonies are counted using a dissectingmicroscope.

Example 46: Tumorigenicity Assay In Vivo

Terminally-differentiated cultured erythroid cells are implanted invarious animal models to evaluate the potential for tumorigenicity.Several tissues are collected from the various models and analyzed withhistological, immunochemical, and fluorescent assays to quantifytumorigenicity.

Animals receive daily intraperitoneal injections of CsA (10 mg/kg,Sandimmune, Novatis Pharma, Nurnberg) starting two days before grafting.For the depletion of NK cells, some rats receive, in addition to CsAintraperitoneal injections of the monoclonal antibody (mAb), anti-NKR-P1A (clone 10/78, mouse IgG₁, BD Biosciences, Heidelberg, Germany) or therespective isotype control (clone PPV-06, mouse IgG₁, Exbio, Prague,Czech Republic). The anti-NKR-P1A mAb (clone 10/78) is directed againstthe same epitope as the mAb (clone 3.2.3). One mg of the respectiveantibodies are given one day before the injection of erythroid cellsfollowed by 0.5 mg at day 4 after cell transplantation.

Blood samples are taken before starting these experiments, at day 0 and4 days after erythroid cell transplantation, and at autopsy (day 92) inorder to determine the proportion of NK cells in the blood by flowcytometry. For the analysis of subcutaneous tumor growth erythroid cellsare injected in 100 μl phosphate-buffered saline (PBS) into the flank ofthe animals. Tumor growth is monitored every second day by palpation andsize is recorded using linear calipers. Animals are sacrificed beforeday 100 when a tumor volume of 1 cm³ in mice and 5 cm³ in rats isreached, when a weight loss of more than 10% occurs, or when anybehavioral signs of pain or suffering are observable. Autopsies of allanimals are performed.

Murine tissue near the site of injection is immediately frozen in liquidnitrogen or placed in phosphate-buffered 4% formalin for 16 h and thenembedded in paraffin. Spleens and lymph nodes are removed for subsequentimmunological analyses. The transplantation of erythroid cells into thestriatum of unilaterally 6-OHDA-lesioned rats is performed. Theseanimals are sacrificed 6 weeks after transplantation.

Animal tissue is analyzed by flow cytometry. Appropriate fluorescent andPE-conjugated antibodies against established cancer cell biomarkers ofCD133, CD3, CD, CD16, CD19, CD20, CD56, CD44, CD24, and CD133 are addedto the excised tissues samples and analyzed to quantify tumorigenicpotential.

Example 47: Deformability by EKTA

Erythroid cells cultured as described herein are assessed fordeformability characteristics relative to natural erythrocyte samplesvia ektacytometry.

The ektacytometer consists of a Couette-type viscometer combined with ahelium-neon laser used to produce a diffraction image of red cellssuspended in a viscous fluid between the two cylinders. When theviscometer rotates, normal red cells elongate in the shear field,causing the diffraction image to become elliptical. The ellipticity ofthe image is measured by quantifying the light intensity along the major(A) and minor (B) axes of the diffraction pattern and expressing this asa ratio (A−B)/(A+B), the deformability index (DI) or elongation index(EI). The viscosity of the medium is chosen to be greater than theinternal viscosity of the densest erythroid cells. A 31 g/liter solutionof polyvinylpyrrolidone (PVP), mw=360,000, in a phosphate buffer of 0.04M composed of K2HPO4 and KH2P04 in distilled water yields a viscosity of0.20 poise at 25° C. and 12 poise at 37 C.

Osmolarity is adjusted with NaCl to the desired level and measured in aRoebling freezing-point osmometer. The final pH is varied by using smalladditions of 1-M solutions of NaOH and HCl and is measured in aTechnicon BG I1 blood gas analyzer. Sodium azide is added as apreservative to stock solutions to obtain 0.4 g/l.

The ektacytometer collects three primary metrics from the erythroid cellsamples and compares them to native erythrocytes; Osmolality minimum(O_(min)), deformability index (Di_(max)), and the osmolality at whichthe DI reaches half of its maximum value (O_(hyp)).

O_(min) is related to the surface area to volume ratio of the cell andhas been found to equal the 50% hemolysis point in the classical osmoticfragility test.

Di_(max) is the maximum value of the deformability index, normallyreached at 290 mosmol (the physiologic osmolality value). This indicatesthe maximum deformability of the cell under shear stress and is relatedto a number of factors, such as surface area, volume, internalviscosity, and mechanical properties of the cell membrane.

O_(h) is the osmolality at which the DI reaches half of its maximumvalue. This gives an indication of the position of the hypertonic partof the curve, which is related to the internal viscosity of the cell aswell as mechanical properties of the membrane, such as how it will bendunder force (stiffness).

The parameters obtained for the cultured erythroid cells are compared tothe same values for primary erythroid cells.

Example 48: Deformability by LORCA

The deformability of purified cRBC populations is measured by a laserdiffraction technique (LORCA, laser-assisted optical rotational cellanalyzer, R&R Mechanotrics). In brief, a highly diluted suspension ofcells is sheared in a Couette system with a gap of 0.3 mm between 2cylinders, one of which is able to rotate to induce shear stresses. Alaser beam is passed through the suspension, and the diffraction patternis measured at 37° C. At low shear stress, the cells are circular disks,whereas at high shear stress, the cells become elliptical. The celldeformability is expressed in terms of the elongation index (EI), whichdepends on the ellipticity of the deforming cells. Aliquots containing12.5 uL of pelleted RBC pellets are diluted in 5 mL ofpolyvinylpyrrolidone solution (molecular weight 360 000). The EI valuesat 30 Pa (referred to as EImax) and 3 Pa are selected as representativevalues of the deformability for easy comparison between samples atvarious shear stresses.

Example 49: Assessment of Vascular Occlusion—Ex Vivo Rat Vasculature

The potential for vascular occlusion of erythroid cells can be assessedwith isolated artificially perfused rat vasculature using methods knownin the art, see e.g., Kaul et al. 1983, J Clin Invest 72:22. Briefly, inanesthetized (sodium pentabarbitol 30 mg/kg) rats of the Wistar strain,120-150 g, the right ileocolic artery and vein are cannulated withheparinized (100 uL/mL) silastic tubing at a site 3 cm distant from theileocolic junction. Under a steady-state perfusion with Ringer'scontaining 1% bovine serum albumin, the ascending colon and terminalileum (3 cm each) are sectioned between ties. After hemostatic ties ofall vascular connections is achieved, the tissue is isolated. Theisolated mesoappendix is gently spread on an optically clear Luciteblock on a microscope stage. The entire preparation is covered with aplastic saran wrap except for outlets of cannulas and the microscopeobjective.

The control arterial perfusion pressure (Ppa) and venous outflowpressures (Pv) are kept constant at 80 and 3.8 mmHg, respectively, andmonitored via Statham-Gould P-50 pressure transducers (StathanInstruments Inc, Oxnard Calif.). The venous outflow (Fv) rate ismonitored using a photoelectric dropcounter and expressed in mL/min Alapse of 10-12 min is allowed for tissue equilibration and stabilizationof Fv. Only preparations exhibiting mesoappendix microvasculature freeof host blood cells and with a steady Fv of 4.6+/−0.5 (mean+/−SD) areused. The experiments are done at 37 C.

Erythroid cells are isolated as described herein. After controlmeasurements of Ppa and Fv, erythroid cells (0.2 mL, Hct 30%) are gentlydelivered via an injection port 15 cm distal to site of arterialcannulation, and the changes in Ppa and Fv are recorded on the stripchart of a Grass polygraph (Grass Instrument Co, Quincy Mass.). Thetissue preparations are perfused for 10-15 min before the infusion ofsamples with Ringer's solution to allow stabilization of the tissue andclear the vasculature of the remaining blood cells of the host animal.The resulting obstruction after the infusion of cells can be cleared andthe flow restored by briefly (2-3 min) perfusing the vasculature withfully-oxygenated Ringer's solution at high pressure (100 mmHg).

At the end of each experiment the entire tissue preparation (free ofcannulas and luminal content) is weighed. Peripheral resistance units(PRU) are calculated and expressed as PRU=ΔP/Q=mmHg/mL/(min-g) where ΔP(mmHg) is the arteriovenous pressure difference and Q (mL/min-g) is therate of venous outflow per gram of tissue.

In each experiment, pressure-flow recovery time (Tpf) is determinedfollowing the bolus infusion of samples. Tpf is defined as the time(seconds) required for Ppa and Fv to return to their base-line levelsfollowing the delivery of a given sample, and it represents totaltransit time throughout the mesoappendix vasculature. The parametervalues obtained for cultured erythroid cells are compared to the valuesobtained for primary erythroid cells.

Example 50: Assessment of Vascular Occlusion—In Vitro Flow Chamber

Methods to assess vascular occlusion of erythroid cells using in vitrograduated height flow chambers are known in the art, see e.g., Zennadiet al 2004, Blood 104(12):3774.

Briefly, graduated height flow chambers are used to quantitate theadhesion of erythroid cells to endothelial cells (ECs). Slides coatedwith ECs are washed with Hanks balanced saline solution (HBSS) with 1.26mM Ca2+, 0.9 mM Mg2+ (Gibco, Grand Island, N.Y.) warmed previously to37° C. and then fit into a variable height flow chamber. The flowchamber is mounted on the stage of an inverted phase contrast microscope(Diaphot; Nikon, Melville, N.Y.) connected to a thermoplate (Tokai Hit,Fujinomiya-shi, Japan) set at 37° C. Cells are observed using a videocamera (RS Photometrics, Tucson, Ariz.) attached to the microscope andconnected to a Macintosh G4 computer (Apple, Cupertino, Calif.).Erythroid cells are cultured as described herein, and labeled withfluorescent dye PKH 26 red fluorescent cell linker kit (Sigma) followingthe manufacturer's instructions. Cells (3 mL) suspended at 0.2%(vol/vol) in HBSS with Ca2+, Mg2+ are infused into the flow chamber andallowed to adhere to the slide for 15 minutes without flow. Beforeexposure to flow, a minimum of 3 fields at each of 7 different locationsalong a line oriented normal to future flow are examined for the totalnumber of fluorescent cells. Fluid flow (HBSS with Ca2+, Mg2+) is thenstarted using a calibrated syringe pump. After exposure to flow, thefields are again examined and the number of adherent cells counted. Thefraction of adherent cells is presented as follows: Number of cellsattached after exposure to flow/Cells present per field before flow. Thewall shear stress is calculated as follows: τw=(6μQ)/(wH [x]2), in whichτw indicates wall shear stress (dyne/cm2); Q, volumetric flow rate(cm3/s); μ, media viscosity; w, width of the flow channel; and H(x),height of the flow chamber as a function of position along themicroscope slide.

Example 51: Assessment of Vascular Occlusion—Intravital Microscopy

Methods to assess vascular occlusion of erythroid cells using intravitalmicroscopy are known in the art, see e.g., Zennadi et al. 2007 Blood110(7):2708.

Briefly, general anesthesia of a test animal is achieved byintraperitoneal injection of 100 mg/kg ketamine (Abbott Laboratory,Chicago, Ill.) and 10 mg/kg xylazine (Bayer, Shawnee Mission, Kans.). Adouble-sided titanium frame window chamber is surgically implanted intothe dorsal skin fold under sterile conditions using a laminar flow hood.Surgery involves carefully removing the epidermal and dermal layers ofone side of a dorsal skin fold, exposing the blood vessels of thesubcutaneous tissue adjacent to the striated muscles of the opposingskin fold, and then securing the 2 sides of the chamber to the skinusing stainless steel screws and sutures. A glass window is placed inthe chamber to cover the exposed tissue and secured with a snap ring.Subsequently, animals are kept at 32° C. to 34° C. until in vivo studieswere performed 3 days after surgery.

Anesthetized animals with window chambers are placed on the stage of anAxoplan microscope (Carl Zeiss, Thornwood, N.Y.); temperature ismaintained at 37° C. using a thermostatically controlled heating pad.All infusions are through the dorsal tail vein. Erythroid cells arecultured as described herein. Cells are then labeled with Dil or DiO(Molecular Probes, Eugene, Oreg.) dyes per manufacturer's instructions.Labeled cells (300 μL; hematocrit 0.50 [50%] in PBS with Ca2+ and Mg2+)are infused, and RBC adhesion and blood flow dynamics are observed insubdermal vessels for at least 30 minutes using LD Achroplan 20×/0.40Korr and Fluar 5×/0.25 objectives. Microcirculatory events and celladhesion are simultaneously recorded using a Trinitron Color videomonitor (PVM-1353 MD; Sony, Tokyo, Japan) and JVC video cassetterecorder (BR-S3784; VCR King, Durham, N.C.) connected to a digital videocamera C2400 (Hamamatsu Photonics KK, Hamamatsu City, Japan). Thirtysegments of venules are examined for each set of conditions. Arteriolesare distinguished from venules based on (1) observation of divergentflow as opposed to convergent flow; (2) birefringent appearance ofvessel walls using transillumination, which is characteristic ofarteriolar vascular smooth muscle; and (3) relatively straight vesseltrajectory without evidence of tortuosity.

Measurement of red cell flux and adhesion is performed by examiningvideotapes produced using×20 magnification. Cell adherence isquantitated by considering cells attached to the vessel walls andimmobile for 1 minute. The percentage of the length of vessels withdiameters up to 25 μm or more than 25 μm, occupied by SS RBCs, isquantified as follows: % venular length occupied by SS RBCs=(length ofvessel wall with adherent cells/total length of the vessel segmentsanalyzed)×100. Changes in RBC flux are calculated as follows:flux=number of circulating fluorescent human RBCs crossing a singlepoint marked on vessels less than 50 μm in diameter per minute.

Example 52: Assessment of Vascular Occlusion—Platelets

Methods to assess vascular occlusion of platelets using human vascularendothelial cells (HUVECs) can be adapted from similar methods foreythroctyes. Briefly, a 2-mL volume of 0.05% hematocrit suspension isadded to confluent HUVECs on tissue culture Petri dish. Thecone-and-plate apparatus is assembled within 1 min after addition ofplatelets and placed on a Nikon Diaphot-TMD inverted-phase contrastmicroscope (Southern Micro Instruments, Atlanta, Ga.). The motor isstarted to turn the cone, and adherence is continuously monitored at 0.1or 1 dyne/cm2 shear stress for 30 min Temperature is maintained constantat 37° C. by an air curtain incubator (Nicholson Precision Instruments,Inc., Bethesda, Md.) blowing on the adhesion apparatus. Plateletadherence is visualized and recorded every 5 min by focusing on 8different fields of view for 20 sec per field for each time point. Theentire experiment is viewed under 400× total magnification through aCCD-72 series camera (Dage-MTI, Inc., Michigan City, Ind.) and recordedon videotape with a SVO 2000 video cassette recorder (Sony Electronics,San Jose, Calif.). Adherence is quantified off-line at the end of eachexperiment by counting individual adherent cells during manual playbackof recorded video images. The cell counts in 8 fields for each timepoint are averaged and normalized to adherent red cells per squaremillimeter of endothelium.

Example 53: Assessment of Mass/Volume/Density with Resonator

A dual suspended microchannel resonator (SMR) system is used tocharacterize the mass, volume, and density of a population ofterminally-differentiated erythroid cells based on Bryan et al, LabChip,2014. At the start of a cell density measurement, the system is firstflushed with filtered Percoll media, which serves as the high densityfluid. Next, the sample bypass is filled with a dilute cell sample, andthe vial heights at the sample inlet and outlet are adjusted to directfluid flow into the first SMR. Pressure at the high density fluid inletis used to set the density of Fluid 2, and pressure at the waste outletcontrols the overall flow speed in the device. To minimize thelikelihood of size biasing due to heavier cells settling at the bottomof the sample vial or tubing, a fresh sample is introduced at regularintervals by flushing the sample bypass channel Data is acquired viaLabVIEW and processed with MATLAB.

Cell concentration is monitored using a Coulter counter. Cellmeasurements are performed on cultures grown to 5×10̂5-1×10̂6 cells/ml.High density fluid introduced for measurement in the second SMR isformulated as a solution of 50% (v/v) Percoll (Sigma), 1.38% (w/v)powdered L15 media (Sigma), 0.4% (w/v) glucose, 100 IU penicillin, and100 μg mL—1 streptomycin. Media pH is adjusted to 7.2. This Percollmedia is stored at 4° C. and filtered immediately prior to use in thedual SMR.

Example 54: Assessment of Phosphatidyl Serine Content by Annexin V

Erythroid cells are cultured as described herein. 50 μL cell suspensionis washed in Ringer solution containing 5 mM CaCl₂ and then stained withAnnexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) inthis solution at 37° C. for 20 min under protection from light. Cellsare washed and stained by flow cytometry as described herein, andannexin-V fluorescence intensity is measured with an excitationwavelength of 488 nm and an emission wavelength of 530 nm. Relativephosphatidyl serine exposure is assessed from annexin-V fluorescence.

Example 55: Assessment of Lipid Content by Chromatography

Lipids are extracted from washed synthetic membrane-receiver complexesby three extractions with methanol-chloroform 1:1 at room temperature inthe presence of the antioxidant BHT (Sigma Aldrich). The pooled extractsare washed with 0.05 M KCl in the method of Folch, Lees and SloaneStanley 1957, J Biol Chem 226:497. Briefly, for the first extraction, 15mL methanol containing 0.05 mg/mL BHT are added to the washed complexesin a centrifuge tube and allowed to stand for 30 min with occasionalstirring to break up sediment. 15 mL of chloroform is then added and themixture is allowed to stand for 30 min with occasional stirring to breakup clumps. The tubes are centrifuged for 5 minutes at 1500 g and thesupernatant fractions decanted into separatory funnels fitted withTeflon stopcocks. The second and third extractions are performedsimilarly with 15 mL of the methanol-BHT added to the residue followedby 15 mL of chloroform, except the extracts stand for only 10 minuteswith occasional stirring after each addition. After centrifugation, thesupernatant fractions are pooled in a separatory funnel then 48 mL ofchloroform and 28 mL of 0.05 M KCl are added and mixed. The mixture isallowed to stand overnight in darkness at 4 C for phase separation.After being rewarmed to room temperature, the lower of the two clearphases is collected and evaporated to dryness in vacuo at 40 C in arotary vacuum evaporator. The lipid is transferred quantitatively to a10 mL volumetric flask with chloroform and stored at −22 C.

The concentration of free cholesterol in the lipid extract is determinedas follows. The lipid extract is chromatographed on a 0.5 mm layer ofSilica Gel HR (Brinkmann Instruments, Inc., Westbury, N.Y.) inhexane-diethyl ether-glacial acetic acid 80:20:1, the TLC plate isstained by spraying with 2,7-dichlorofluorescein solution (see below),the free cholesterol spot is scraped into a conical centrifuge tube andextracted once with 2.0 ml and three times with 1.0 ml of chloroform,the extract is evaporated to dryness in vacuo at 40° C. in a rotaryvacuum evaporator, and the cholesterol is estimated by the ferricchloride method of Mann 1961 Clin Chem 7:275 without saponification. Afree cholesterol standard, prepared from a commercial certified reagentgrade material by isolation through the dibromide derivative (see e.g.,Fieser J Amer Chem Soc 1953 75:5421), is taken through thechromatographic procedure and estimated with each set of determinations.The values for free cholesterol are corrected in each determination forthe recovery of the standard, which averaged 95%. The TLC is necessaryto remove the BHT, which otherwise interferes with the ferric chloridemethod by producing a brown product that absorbed at 560 nm.

The phospholipid distribution is determined in triplicate by TLC ofaliquots of the total lipid extract at 4° C. on Silica Gel HR, 0.5 mmthick, in chloroformmethanol-glacial acetic acid-water 25:15:4:2 towhich is added BHT at a concentration of 50 mg/100 ml to preventautoxidation during chromatography; the TLC plates are prepared withwater (“neutral” plates). Use of a “wedged-tip technique” for applyingthe lipid sample at the origin of the plate (see e.g., Stahl 1965Thin-Layer Chromatography, Academic Press Inc.) results in excellentseparations of the individual phospholipids. In particular, the methodprovides complete separation between phosphatidyl ethanolamine (PE),phosphatidyl serine (PS), lecithin, and sphingomyelin; a discrete spotmigrates between PS and lecithin that is identified as phosphatidylinositol (PI). The spots are made visible in UV light by spraying with asolution of 2,7-dichlorofluorescein (33.3 mg/100 ml of aqueous 2 mMNaOH) and then scraping directly into Kramer-Gittleman tubes, where thephospholipids are digested at 190° C. for 60 min with 1.0 ml of 70%perchloric acid. The remainder of the procedure is performed asdescribed above, except that after color development, the silica gel isremoved by centrifugation at 3000 g for 5 min and the absorbancy isdetermined on the clear supernatant solution. Corrections are made forthe absorbancy of corresponding areas of blank lanes.

Gas-liquid chromatography is performed on hexane-dissolved samples witha Barber-Colman instrument, model 5000, equipped with paired 8-ftcolumns of EGSS-X (an ethylene glycol succinate polyester combined witha silicone) 8% on Gas-Chrom P, 100-120 mesh (Applied ScienceLaboratories Inc.) and dual flame ionization detectors. The nitrogenflow rate is 50 ml/min at the inlet. The column temperature ismaintained at 1650 C for 10 min after injection of the sample, thenincreased at 2 C/min to 200° C.

Example 56: Assessment of Membrane Viscosity

The membrane viscosity of a population of cells can be assessed byfluorescence photobleaching assay. A 0.5-ml sample of erythroid cells iscollected and washed once in HEPES-buffered saline (132 mM NaCl, 4.7 mMKCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES, adjusted to pH 7.4). Thepacked cells are then washed once in 145 mM NaCl −10 mM NaHCO3, pH 9.5,and resuspended in the same buffer with 1 mg/ml DTAF (obtained fromResearch Organics, Cleveland, Ohio). The cells are incubated on ice for1 h, then washed twice in 50 mM glycine—95 mM NaCl—10 mM NaHCO3, pH 9.5,to remove any dye that has not bound covalently to protein. Finally, thecells are washed twice and resuspended to −2% hematocrit inHEPES-buffered saline with 1 mg/ml bovine serum albumin. The sametreatment is applied to control native erythrocytes.

The flow chamber is mounted on the stage of a Leitz Diavert (Rockleigh,N.J.) inverted microscope equipped for incident-light fluorescencemicroscopy. The dichroic mirror and excitation/emission filters are thestandard combination for use with fluorescein dyes (Leitz designation12), with excitation wavelength in the range 450-490 nm. The objectiveis an oil immersion type with 100× magnification and 1.25 numericalaperture. A 100 watt high pressure mercury arc lamp (Osram, Munich) withan appropriate power supply and housing (Oriel, Stamford, Conn.) servesas the fluorescence excitation source.

A computer-controlled electronic shutter (Vincent Associates, Rochester,N.Y.) limits the exposure duration and is synchronized with aphoton-counting electronic system for measuring fluorescence intensity.The field diaphragm of the incident light illuminator is used to limitexcitation to a circular area of diameter 20-40 um. At regularintervals, an output pulse from the computer causes the shutter to openfor a typical duration of 20 ms. Light from the brief fluorescent imageis split with a series of prisms so that half the light is directed to alow-light-level SIT video camera (Model 66-SIT, Dage-MTI, Michigan City,Ind.) and half to a photomultiplier tube (Model 8850, RCA, Harrison,N.J.) enclosed in an ambient temperature housing. During the time thatthe electronic shutter is open, a video image processor (Model 794,Hughes Aircraft, Carlsbad, Calif.) is triggered to acquire thefluorescent image, providing a video snapshot that can be monitored toensure that the subject remains in focus and that no foreign objectintrudes into the field of view. Distances on the video screen aremeasured with a video caliper and calibrated by comparison with thevideo image of a stage micrometer. Also during the time the shutter isopen, the photomultiplier signal is processed with the photon-countingtechnique. An amplifier/discriminator (Model AD6, Pacific Instruments,Concord, Calif.) generates a digital logic pulse for each signal pulseabove a given magnitude, and those digital pulses are counted on a100-MHz gated counter (Model 770, EG&G Ortec, Oak Ridge, Tenn.). Themicrocomputer controls the gating, resetting, and recording of thephoton count.

A typical experiment consists of a number of preliminary fluorescencemeasurements made during brief (20 ms) pulses of excitation light,followed by an extended period of illumination (typically 30 s) duringwhich the samples cells are bleached, followed by another series ofbrief exposures, every 15-30 s, until the fluorescence appears to havecompleted its recovery.

The recovery time and other parameter values obtained for culturederythroid cells are compared to the same values obtained for primaryerythroid cells.

Example 57: Assessment of Mean Corpuscular Volume with Advia HematologyAnalyzer

The Mean corpuscular volume (MCV) of the cultured erythroid cells ismeasured using electrical impedance in an Advia 120 hematology analyzer(Siemens Healthcare). The results are compared to that of natural humanerythrocytes.

Example 58: Pathogen Testing of Cultured Erythroid Cells

RT-PCR is used to quantify adventitious virus presence in culturederythroid cell populations and confirm non-contamination (Assay No.003000.BSV, BioReliance). Sterility testing of unprocessed and finalbulk, final vials, prebanking cells, and cell and virus banks isperformed by directly inoculating the erythroid population into 2different types of media that support the growth of aerobic andanaerobic bacteria respectively. Samples are incubated for 14 daysfollowed by testing for microbial contaminants per BioReliance SterilityTesting protocol USP 71.

Example 59: Assessment of Osmotic Fragility

Osmotic fragility is evaluated to measure the resistance of theerythroid cells to lysis when exposed to hypotonic solutions. Solutionsof NaCl in water were made at concentrations spanning 0% to 1%. Cellswere incubated in each of the salt solutions for 15 minutes. The sampleswere centrifuged to pellet intact cells. Supernatant was assayed forhemoglobin content by absorption of light at 540 nm using aspectrophotometer. The point at which 50% hemolysis occurs is calculatedand compared to the value obtained for primary erythrocytes.

Example 60: Assessment of Rosetting/Immunogenicity

The direct antiglobulin test, also known as Coombs test, assesses theagglutination or resetting of erythroid cells caused by the binding ofpolyclonal antibodies from serum to surface antigens on the cell. It canbe performed with pooled human serum for general allogeneicimmunogenicity assessment, or with serum from the intended recipient forspecific immunogenicity prediction.

In brief, add 1-2 drops of cells stored in an EDTA tube to a reactiontube. Wash this tube three times with isotonic saline. After the thirdwash, prepare a 3% suspension from the washed cells. Label 2 tubes A andB. Add one drop of the washed 3% suspension to each tube. Wash thesetubes one more time. When decanting, position the tubes so that the cellbutton is on top. This will prevent too many cells from being lost inthe washing process. Drain well, and blot dry with a biowipe Immediatelyadd one drop human test serum to both tubes, and shake to mix. Allow theB tube to incubate at room temperature 5 minutes. Centrifuge the A tubefor the time calibrated for the Coombs spin on the serofuge. Immediatelyresuspend gently and examine for agglutination using the lightedagglutination viewer (Beckton Dickinson). If the A tube is positive, itis not necessary to read the B tube nor is it necessary to examine the Atube microscopically. If the A tube is negative by lighted agglutinationviewer, examine for agglutination under the microscope. If the A tubewas negative through the microscopic reading, centrifuge the B tubeafter its incubation period and repeat steps 2-4 with the B tube sample.If the B tube is negative as well, add one drop of IgG-coated CoombsControl Cells (Check Cells) to the tube and centrifuge. Examine foragglutination. Agglutination should be present in this step, or the testis invalid.

If there is no agglutination in any of the steps before addition of thecheck cells (ccc), the test is interpreted as negative. If agglutinationis observed in any of the steps before addition of the check cells, thetest is interpreted as positive.

Example 61: Assessment of Oxygen-Binding Capacity

Equilibrium oxygen binding curves at 37° C. are determined in atonometer linked to a 1-cm path length cuvette. Spectral measurementsare performed with a spectrophotometer (Cary 50; Variant Inc), and thetemperature is controlled with a Peltier module. Analyses are performedin 50 mM bis-Tris buffer (pH 7.2) containing 140 mM NaCl and 2 mMglucose. After thorough deoxygenation under nitrogen, the red cellsuspensions are equilibrated at different partial pressures of oxygen byinjection of known volumes of pure oxygen into the tonometer through arubber cap with a Hamilton syringe. The fractional saturation isestimated by simulation of the absorption spectra in the visible andSoret regions as a linear combination of the fully deoxygenated andoxygenated spectra of the RBC suspension by least squares regression.

Example 62: Assessment of Metabolic State of Cells

The erythroid cell population may be verified as metabolically activeusing a variety of different enzyme based assays to quantify importantmetabolic end products. Active glycolysis is a crucial metabolic pathwayto assess and may be measured with the following assay (Glycolysiscell-based assay kit, Cayman Chemical, Item 600450).

450 ul of assay buffer is aliquoted into a test tube, followed by 50 uLof the L-Lactic acid standard asnd mixed thoroughly. A titration curveis constructed using the lactic acid concentration standard, beginningwith a 1 mM dilution.

Cells are added to a 96 well plate and centrifuged at 1000 RPM for 5minutes. 100 uL of the standards are transferred into a separate 96 wellplate. 90 uL of assay buffer is then added to each well. 10 ul ofsupernatant in each cell well is then transferred to corresponding newwells. Add 100 ul of reaction solution to each well using a repeatingpipettor. The plates are then incubated on an orbital shaker for 30minutes at RT. The absorbance isread at 490 nm with a plate reader.Results are compared to natural cells to identify any metabolicdifferences.

Example 63: Assessment of Platelet Aggregation

Aggregation propensity of cultured or primary sourced platelets can bemonitored. Platelets are submitted to swirling analysis by shaking themin front of a light source, with the results expressed as presence orabsence of birefringence. The units of platelet concentrates producedwith a volume of 50-70 mL are left to rest for one hour and placed in alinear shaker (C-Mar®) at 70 rpm at a controlled temperature of 22±2° C.(71.6±3.6° F.).

The tests of platelets concentrates (platelet count, plateletaggregation and pH) are carried out on days 1, 3 and 5 after processing;a leukocyte count is performed only on day 1 and the microbiologicalcontrol is performed only on the 5th day of storage. In order to obtainaliquots from samples of platelet concentrates, a sterile connection(Haemonetics®) is used which ensured the integrity of the environment.Platelet aggregation is achieved using the turbidimetric aggregometrytechnique using a dual-channel Chronolog (Crono-Log Corporation®) withinfour hours of blood collection. For this, the cells are initiallyobtained through light centrifugation at 1000 rpm for five minutes, andthen centrifuged at 3000 rpm for fifteen minutes (Eppendorf®). Samplesare subjected to a platelet count in an automatic counter (HumanCount®).

After adjusting the platelet concentration, aggregation is evaluatedusing different concentrations of inducing agonists: collagen 2.0 μg/mLand ADP 7.0 μg/mL (Crono-Log Corporation®). For each test, 400 μL of PRPand 400 μL of PPP are used, each one in a different cuvette afterwaiting for spontaneous aggregation. The aggregation curve is observedafter five minutes of stimulation by inducing agonists, and soon after,aggregation is measured and expressed as a percentage according to thecurves formed during the tests. The result of the test is commonlyexpressed as a percentage of aggregation by the quantity of lighttransmitted through the test solution; aggregation is classified asnormal, low or high.

Example 64: Autologous Culture Process

The culture of erythroid cells using autologously sourced progenitorCD34+ cells is done to optimize cell immunocompatibility for patients.CD34+ cells from the bone marrow are mobilized to the periphery in apatient using GM-CSF as described herein. Between 10̂6-10̂8 CD34+ cellsare collected and cultured using the aforementioned 22 day protocolusing defined media. During Day 4 the cells are transfected with alentiviral vector containing a gene that codes for the expression of atherapeutic agent. Upon completion of the culturing protocol, the cellsare purified and assessed across several quality control metricsincluding physical properties that correlate with circulation viability,immunogenicity, replicative potential, purity, and therapeutic dose. Thecells are then stored in appropriate stabilizing solution and formulatedin a syringe or appropriate delivery vehicle. The cells are then infusedinto the same patient that donated the initial CD34+ cells.

Example 65: Autologous Loading Process

For the preparation of therapeutic erythroid cells loaded with asuitable receiver, autologously sourced erythrocytes can be used tooptimize cell immunocompatibility for patients. Blood is drawn from thepatient and centrifuged at 5000 g for 20 minutes. The buffy coat isremoved and the remaining red cells are re-suspended in anticoagulantbuffer at a density of 10̂8 cells/ml, giving a total of 10̂10 cells. Thecells are loaded with a therapeutic receiver of interest by one of themethods described above. Upon completion of the loading protocol, thecells are purified and assessed across several quality control metricsincluding physical properties that correlate with circulation viability,immunogenicity, replicative potential, purity, and therapeutic dose. Thecells are then stored in appropriate stabilizing solution and formulatedin a syringe or appropriate delivery vehicle. The cells are infused intothe same patient that donated the initial erythrocytes.

Example 66: Allogeneic Culture Process

To create a scalable, universal therapeutic, etyrhoid cells can becultured from an allogeneic source. The culture of erythroid cells usingallogeneically sourced progenitor CD34+ cells is done to streamline theprocess and culture a volume of therapeutic capable of treating patientsat scale. Donors are blood-typed for major blood antigens, including A,B, Rh to identify universal donors (e.g., O Rh- or Bombay Rh-). CD34+cells from the bone marrow are mobilized to the periphery in a suitabledonor using GM-CSF as described herein. Between 10̂6-10̂8 CD34+ cells arecollected and cultured using the aforementioned 22 day protocol usingdefined media. During Day 4 the cells are transfected with a lentiviralvector containing a gene that codes for the expression of a therapeuticagent. Upon completion of the culturing protocol, the cells are purifiedand assessed across several quality control metrics including physicalproperties that correlate with circulation viability, immunogenicity,replicative potential, purity, and therapeutic dose. The cells are thenstored in appropriate stabilizing solution and formulated in a syringeor appropriate delivery vehicle. The cells are then infused intopatients irrespective of their major blood groups.

Example 67: Allogeneic Loading Process

The culture of erythroid cells using allogeneically sourced progenitorCD34+ cells is done to streamline the process to prepare larger volumesof therapeutic cells capable of treating patients at scale. Donors areblood-typed for major blood antigens, including A, B, Rh to identifyuniversal donors (e.g., O Rh- or Bombay Rh-). The cells are loaded witha therapeutic receiver of interest by one of the methods describedabove. Upon completion of the loading protocol, the cells are purifiedand assessed across several quality control metrics including physicalproperties that correlate with circulation viability, immunogenicity,replicative potential, purity, and therapeutic dose. The cells are thenstored in appropriate stabilizing solution and formulated in a syringeor appropriate delivery vehicle. The cells are then infused intopatients irrespective of their major blood groups.

Example 68: Storage

1. Storage in Refrigerated Buffer Solution

Standard protocols for the storage of red blood cells are known in theart, see e.g., Meryman and Hornblower 1986, Transfusion 26(6):500. Thestandard protocol for the storage of red blood cells (for up to 42 days)is the collection of blood into anticoagulant solutions(citrate-dextrose-phosphate). Erythroid cells are cultured as describedherein. Red cell concentrates are prepared by the removal of plasma bycentrifugation. The cells are stored at 4±2° C. in a slightly hypertonicadditive solution, SAGM (sodium, adenine, glucose, mannitol, 376mOsm/L).

2. Storage in Frozen Buffer Solution

Methods for glycerolization, freezing, and thawing of erythroid cellsare known in the art, see e.g., Meryman and Hornblower 1977 Transfusion17(5):4348. Human blood in citrate phosphate dextrose is glycerolizedand frozen within 4 days of collection. To prepare glycerolized RBCs,approximately 10 mL of whole blood is first centrifuged at 1,400 g for10-15 min, and the plasma is removed. The resulting packed cells arethen glycerolized in two steps using an aqueous glycerol solution withthe following composition: 57.1 g glycerol, 0.03 g potassium chloride,0.085 g magnesium chloride hexahydrate, 0.08 g disodium phosphate, and1.6 g sodium lactate in a total volume of 100 mL, adjusted to a pH of6.8.42 In the first step, 1.5 mL of this glycerol solution is addeddrop-wise to the packed cells with gentle agitation over a period of 3min. The mixture is then allowed to equilibrate undisturbed for at least5 min. In the second glycerolization step, 5 mL of the glycerol solutionis added drop-wise while the mixture is gently agitated over a 3-minperiod, yielding a final glycerol composition of ˜40% w/v. The entireglycerolization process is carried out at room temperature. Theglycerolized RBCs are then divided into aliquots of 0.6-1.1 mL incryogenic vials, placed in a NalgeneVR Cryo “Mr. Frosty” freezingcontainer (Thermo Scientific, NC), and stored in a −80 C freezer for atleast 12 h and up to 10 years. Frozen RBCs are thawed by placing thecryogenic vial in a 37 C water bath for 1 min. All glycerolized bloodsamples are used in deglycerolization experiments within 2 h of thawing.

3. Formulation as Syringe

The cell population may be intravenously administered via a syringe. Thetherapeutic cells are diluted to a density of 10̂7 cells/ml usingstandard saline buffer at 37 C such that 100 ml of volume, or 10̂9 cells,are delivered. The cell solution is loaded into a 150 cc syringe, 20gauge needle and injected into the patient through the basilic vein at 5cc/min. During injection the patient's vitals are monitored for anyimmunogenic or clotting reactions.

4. Formulation as Bag

The cell population may be intravenously administered via syringeconnected to a bag and drip chamber (i.e. an IV drip). The therapeuticcells are diluted to a density of 10̂7 cells/ml using standard salinebuffer at 37 C such that 100 ml of volume, or 10̂9 cells, are delivered.The cell solution is loaded into a 1 L plastic bag, connected to acatheter and allowed to drain via gravity into the patient through thebasilic vein. During infusion the patient's vitals are monitored for anyimmunogenic or clotting reactions.

Example 69: CAPS Catastrophic Antiphospholipid Syndrome

Cells are cultured in the presence of a lentivirus encoding theexogenous transgene β2-Glycoprotein I (b2GPI) (GenBank: X53595.1) fusedto the N-terminus of glycophorin A such that the final cell productexpresses >1×10̂5 copies of the b2GPI receiver on the surface per cell.To ensure that the receiver is functionally expressed, in vitro activityis assessed by flow cytometry. Briefly, cells are incubated with serumfrom patients with Antiphospholipid Syndrome that have previously testedpositive for anti-b2GPI antibodies by ELISA. The cells are washed andlabeled with secondary antibodies to detect human primary antibodiesbound to their surface and analyzed by flow cytometry for presence ofthe fluorophore.

The cultured erythroid cell population that over expresses beta-2glycoprotein I is provided as a treatment for antiphospholipid syndromein an early phase clinical trial.

A patient diagnosed as having antiphospholipid autoantibodies incirculation is intravenously administered single doses of 10̂9-10̂11 cellsonce a month for 6-12 months. During the course of treatment patients'thymidine and thymine levels are monitored with daily blood tests andrelevant APS symptoms such as thrombotic events and bleeding aredocumented.

A population of 10̂11 erythroid cells expressing between 10K and 100Kcopies of Beta-2 glycoprotein 1 per cell is stored in a transfusion bagwith CPDA-1 and glycerol and stored at −80 C for up to 10 years. Upontreatment, the bag is thawed, centrifuged, and the cells removed andresuspended in saline for administration to a patient. Cells areintravenously administered with a 50 gauge needle at 5 ml/min at 37 C.

Example 70: Goodpasture Disease

Cells are cultured in the presence of a lentivirus encoding thenon-collagenous C-terminal domain of the exogenous transgene COL4A3,NC1-COL4A3 (ID: NM_000091.4) fused to the N-terminus of glycophorin Asuch that the final cell product expresses >1×10̂5 copies of theNC1-COL4A3 receiver on the surface per cell as assessed by flowcytometry. To ensure that the receiver is functionally expressed, invitro activity is assessed by flow cytometry. Briefly, cells areincubated with serum from patients with Goodpasture Syndrome that havepreviously tested positive for anti-NC1-COL4A3 antibodies by ELISA. Thecells are washed and labeled with secondary antibodies to detect humanprimary antibodies bound to their surface and analyzed by flow cytometryfor presence of the fluorophore.

Example 71: Membranous GN

Cells are cultured in the presence of a lentivirus encoding the4^(th)-6^(th) extracellular domains of the exogenous transgenephospholipase A2 receptor (PLA2R) (ID: MGC:178179) fused to theN-terminus of glycophorin A such that the final cell productexpresses >1×10̂5 copies of the PLA2R receiver on the surface per cell asassessed by flow cytometry. To ensure that the receiver is functionallyexpressed, in vitro activity is assessed by flow cytometry. Briefly,cells are incubated with serum from patients with MembranousGlomerulonephritis (MGN) that have previously tested positive foranti-PLA2R antibodies by ELISA. The cells are washed and labeled withsecondary antibodies to detect human primary antibodies bound to theirsurface and analyzed by flow cytometry for presence of the fluorophore.

Example 72: IgA Nephropathy

Cells are cultured in the presence of a lentivirus encoding theextracellular domain of exogenous transgene complement receptor 1 (CR1)(SEQ ID 2) fused to the N terminus of glycophorin A such that the finalcell product expresses >1×10̂5 copies of CR1 ectodomain receiver per cellas assessed by flow cytometry. To ensure that the receiver isfunctionally expressed, the cell is assayed for its ability to bindimmune complexes and transfer those complexes to macrophages.

Dylight 650-labeled bovine serum albumin (BSA-650) is incubated withpolyclonal rabbit anti-BSA (Abcam) in an excess of antibody for 30minutes at room temp to generate complexes. The complexes are then mixedwith human serum at a 1:1 volume ratio for 30 minutes at 37 C to formimmune complexes. Control complexes are either not mixed with humanserum or mixed with heat-inactivated human serum. The complexes are thenincubated with cells for 30 minutes at 37 C. Cells are washed andanalyzed by flow cytometry for capture of immune complexes by detectingDylight 650 fluorescence.

Cultured U937 monocytes are activated by incubation with 100 nM phorbolmyristate acetate (PMA) for 24 hours at 37 C. Cells coated with immunecomplexes (see above) are incubated with activated U937 macrophages for30 minutes at 37 C. The co-culture is analyzed by flow cytometry.Macrophages are identified by FSC/SSC gating. Presence of immune complexon macrophages is analyzed by detecting Dylight 650 fluorescence in thiscell population.

Example 73: Systemic Lupus Erythematosus

Cells are cultured in the presence of a lentivirus encoding theextracellular domain of exogenous transgene complement receptor 1 (CR1)(SEQ ID 2) fused to the N terminus of glycophorin A such that the finalcell product expresses >1×10̂5 copies of CR1 ectodomain receiver per cellas assessed by flow cytometry. To ensure that the receiver isfunctionally expressed, the cell is assayed for its ability to bindimmune complexes and transfer those complexes to macrophages.

Dylight 650-labeled bovine serum albumin (BSA-650) is incubated withpolyclonal rabbit anti-BSA (Abcam) in an excess of antibody for 30minutes at room temp to generate complexes. The complexes are then mixedwith human serum at a 1:1 volume ratio for 30 minutes at 37 C to formimmune complexes. Control complexes are either not mixed with humanserum or mixed with heat-inactivated human serum. The complexes are thenincubated with cells for 30 minutes at 37 C. Cells are washed andanalyzed by flow cytometry for capture of immune complexes by detectingDylight 650 fluorescence.

Cultured U937 monocytes are activated by incubation with 100 nM phorbolmyristate acetate (PMA) for 24 hours at 37 C. Cells coated with immunecomplexes (see above) are incubated with activated U937 macrophages for30 minutes at 37 C. The co-culture is analyzed by flow cytometry.Macrophages are identified by FSC/SSC gating. Presence of immune complexon macrophages is analyzed by detecting Dylight 650 fluorescence in thiscell population.

Example 74: Paroxysmal Nocturnal Hemoglobinuria

Cells are cultured in the presence of a lentivirus encoding theexogenous transgene CD59 (NCBI Reference Sequence: NM_203330.2) with anN-terminal epitope tag such that the final cell product expresses >1×10̂5copies of CD59 receiver per cell as assessed by flow cytometry. Toensure that the receiver is functionally expressed, the cell is assayedfor its ability to inhibit membrane attack complex on sheep erythrocytesin co-culture.

Briefly, fresh sheep erythrocytes in 10% solution of 1× veronal bufferedsaline (VBS) are sensitized with polyclonal rabbit anti-sheep RBCantibody (haemolysin) for 30 minutes at 30 C. Serial dilutions ofcultured cells are added to sensitized sheep erythrocytes. Human serumis added at serial dilutions to each well, starting with 1:4 dilution inVBS. Incubate at 37° C. for 30 minutes, mixing after 15 minutes.Centrifuge the samples at 1,500 g for 5 minutes to sediment the RBCs.Transfer 100 ul of supernatant from each tube to a well in a 96 wellflat bottom plate. Add 100 ml of distilled water to each well. Read theabsorbance of the samples at 540 nm using a plate spectrophotometer. %lysis is calculated as OD540 (test)−OD540 (blank)/OD540 (totallysis)−OD540 (blank)*100.

Example 75: Atypical Hemolytic Uremic Syndrome

Cells are cultured in the presence of a lentivirus encoding theexogenous transgene CD59 (NCBI Reference Sequence: NM_203330.2) with anN-terminal epitope tag such that the final cell product expresses >1×10̂5copies of CD59 receiver per cell as assessed by flow cytometry. Toensure that the receiver is functionally expressed, the cell is assayedfor its ability to inhibit membrane attack complex on sheep erythrocytesin co-culture.

Briefly, fresh sheep erythrocytes in 10% solution of 1× veronal bufferedsaline (VBS) are sensitized with polyclonal rabbit anti-sheep RBCantibody (haemolysin) for 30 minutes at 30 C. Serial dilutions ofcultured cells are added to sensitized sheep erythrocytes. Human serumis added at serial dilutions to each well, starting with 1:4 dilution inVBS. Incubate at 37° C. for 30 minutes, mixing after 15 minutes.Centrifuge the samples at 1,500 g for 5 minutes to sediment the RBCs.Transfer 100 ul of supernatant from each tube to a well in a 96 wellflat bottom plate. Add 100 ml of distilled water to each well. Read theabsorbance of the samples at 540 nm using a plate spectrophotometer. %lysis is calculated as OD540 (test)−OD540 (blank)/OD540 (totallysis)−OD540 (blank)*100.

Example 76: Age-Related Macular Degeneration

Cells are cultured in the presence of a lentivirus encoding theexogenous transgene CD59 (NCBI Reference Sequence: NM_203330.2) with anN-terminal epitope tag such that the final cell product expresses >1×10̂5copies of CD59 receiver per cell as assessed by flow cytometry. Toensure that the receiver is functionally expressed, the cell is assayedfor its ability to inhibit membrane attack complex on sheep erythrocytesin co-culture.

Briefly, fresh sheep erythrocytes in 10% solution of 1× veronal bufferedsaline (VBS) are sensitized with polyclonal rabbit anti-sheep RBCantibody (haemolysin) for 30 minutes at 30 C. Serial dilutions ofcultured cells are added to sensitized sheep erythrocytes. Human serumis added at serial dilutions to each well, starting with 1:4 dilution inVBS. Incubate at 37° C. for 30 minutes, mixing after 15 minutes.Centrifuge the samples at 1,500 g for 5 minutes to sediment the RBCs.Transfer 100 ul of supernatant from each tube to a well in a 96 wellflat bottom plate. Add 100 ml of distilled water to each well. Read theabsorbance of the samples at 540 nm using a plate spectrophotometer. %lysis is calculated as OD540 (test)−OD540 (blank)/OD540 (totallysis)−OD540 (blank)*100.

Example 77: B Cell Acute Lymphoblastic Leukemia

An antibody scFv is generated based on a full-length anti-CD20 antibody.Splice overlap extension PCR (SOE-PCR) are used to create fullysynthetic anti-CD20 variable (V) genes based on the V gene sequences ofthe murine 2B8 (U.S. Pat. No. 5,736,137). Full-length 2B8 VL and VHgenes are then assembled by SOE-PCR to produce a single chain Fv (scFv)with 18-residue long linker (Whitlow 218 linker; GSTSGSGKPGSGEGSTKG (SEQID NO: 30)) in VL-VH orientation. Following SOE-PCR which also includesa signal peptide to the 5′-end (upstream) to enable secretion, theconstruct is cloned into pCR®-2.1-TOPO vector (Invitrogen Corp.,Carlsbad, Calif.) and confirmed by sequencing.

Cells are cultured in the presence of a lentivirus encoding theexogenenous anti-CD20 antibody scFv anti-CD20 (Olafsen et al., J NuclMed 2009, 50(9):1500) fused to the N-terminus of glycophorin A such thatthe final cell product expresses >1×10̂5 copies of anti-CD20 scFvreceiver per cell as assessed by flow cytometry. To ensure that thereceiver is functionally expressed, in vitro activity is assessed byflow cytometry. Briefly, cells are incubated with soluble CD20 targetprotein (Abcam) at a range of concentrations. The target proteins aredirectly labeled with a fluorophore. Incubated cells are washed andanalyzed by flow cytometry for presence of the fluorophore.

Example 78: Light Chain Amyloidosis

Cells are cultured in the presence of a lentivirus encoding theexogenous transgene Serum Amyloid P (SAP) component (GenBank: D00097.1)fused to the N terminus of glycophorin A such that the final cellproduct expresses >1×10̂5 copies of SAP receiver per cell as assessed byflow cytometry. To ensure that the receiver is functionally expressed,in vitro activity is assessed by flow cytometry. Briefly, cells areincubated with serum from patients with light chain amyloidosis that arepositive for amyloid plaques by anti-Light Chain ELISA. Binding of lightchain amyloid plaques to the SAP-displaying cells is detected bysecondary labeling with anti-lambda light chain antibodies (Abcam) thatare directly labeled with fluorophore. Incubated cells are washed andanalyzed by flow cytometry for presence of the fluorophore.

Example 79: Hepatitis B

Cells are cultured in the presence of a lentivirus encoding theexogenous transgene antibody scFv against hepatitis B surface antigen(HBsAg) (SEQ ID No. 1) such that the final cell product expresses >1×10̂5copies of antibody scFv receiver per cell as assessed by flow cytometry.To ensure that the receiver is functionally expressed, in vitro activityis assessed by flow cytometry. Briefly, cells are incubated with targetprotein HBsAg (Abcam) that is directly labeled with Dylight 650fluorophore at a range of concentrations. Incubated cells are washed andanalyzed by flow cytometry for presence of the fluorophore.

Example 80: ADA-SCID

Adenosine deaminase activity is monitored in vitro using HPLC protocolto detect adenosine and inosine levels. Approximately 10̂5 erythroidcells expressing an exogenous, intracellular adenosine deaminase,produced using the aforementioned transfection protocol, are aliquotedinto 1 ml wells. 1 mM of adenosine is administered to each well andincubated for 1 hr. The cells are centrifuged and soluble protein isremoved from the supernatant using cold methanol precipitation. Thesupernatant samples are then run on an Agilent 1100 HPLC using astandard inosine and adenosine curve to determine the relative amountsof nucleoside and intracellular enzymatic activity compared to naturalcells.

Adenosine deaminase activity is monitored in vivo using HPLC protocol todetect adenosine and inosine levels. An ADA-SCID mouse model is treatedwith clodronate for 3 days prior to cell therapy administration. 100 ulof 10̂8 ADA expressing human erythroid cells are administered via tailvein injection to the mouse model and blood samples are taken via asubmandibular bleed at 10 min, 12 h, 24, h, 48 h, 72 h. The samples areanalyzed using HPLC and inosine and adenosine levels are tracked overtime.

A population of 10̂8 cultured erythroid cells expressing 10K to 100Kcopies of ADA per cell is administered via tail vein injection to acohort of NOD-SCID mice. Prior to injection adenosine and inosinecirculation levels are documented. Cells are allowed to circulated for 1week and blood samples are taken at 10 min, 1 h, 6, h, 12 h, 24, h, 48h, 96 h, 144 h. Adenosine and inosine levels are tracked.

A patient diagnosed with ADA-SCID is confirmed via genotyping and foundto be deficient for ADA activity. Relevant clinical symptoms used in thediagnosis include lymphocyte count, adenosine levels, and infectionfrequency. The patient is intravenously administered 10̂11 erythroidcells cultured from a blood-type matched donor and expressing exogenousADA diluted in 500 ml of saline solution via gravity drain over thecourse of 1 hr. The procedure is repeated monthly for 6 months. Patientlymphocyte counts are tracked using immunofluorescence analysis ofspecific CD antigens, adenosine and inosine levels are monitored usingHPLC, and infection rate recorded over the duration of the treatment.Immunogenic response to the transfusion is closely monitored.

The cultured erythroid cell population that over expresses adenosinedeaminase is provided as a treatment for ADA-SCID.

A patient diagnosed as deficient for adenosine deaminase isintravenously administered single doses of 10̂9-10̂11 cells once a monthfor 6-12 months. During the course of treatment patients' adenosine andinosine levels are monitored with daily blood tests and relevantADA-SCID symptoms such as lymphocyte counts, infections, and skin rashesare documented.

A population of 10̂11 erythroid cells expressing between 10K and 100Kcopies of ADA per cell is stored in a transfusion bag with CPDA-1 andglycerol and stored at −80 C for up to 10 years. Upon treatment, the bagis thawed, centrifuged, and the cells removed and resuspended in salinefor administration to a patient. Cells are intravenously administeredwith a 50 gauge needle at 5 ml/min at 37 C.

Example 81: MNGIE

Thymidine phosphorylase (TP) activity is monitored in vitro using HPLCprotocol to detect thymidine and thymine levels. Approximately 10̂5erythroid cells expressing an exogenous, intracellular thymidinephosphorylase, produced using the aforementioned transfection protocol,are aliquoted into 1 ml wells. 1 mM of thymidine is administered to eachwell and incubated for 1 hr. The cells are centrifuged and solubleprotein is removed from the supernatant using cold methanolprecipitation. The supernatant samples are then run on an Agilent 1100HPLC using a standard thymidine and thymine curve to determine therelative amounts of nucleoside and intracellular enzymatic activitycompared to natural cells.

Thymidine phosphorylase activity is monitored in vivo using HPLCprotocol to detect thymidine and thymine levels. A TP deficient mousemodel is treated with clodronate for 3 days prior to cell therapyadministration (Haragushi, Mol. Cell Biol 2002). 100 ul of 10̂8 TPexpressing erythroid cells are administered via tail vein injection tothe mouse model and blood samples are taken via a submandibular bleed at10 min, 12 h, 24, h, 48 h, 72 h. The samples are analyzed using HPLC andthymidine and thymine levels are tracked over time.

A patient diagnosed with MNGIE is confirmed via genotyping and found tobe deficient for TYMP. Relevant clinical symptoms used in the diagnosisinclude gastrointestinal motility, early satiety, cachexia, and nausea.The patient is intravenously administered 10̂11 erythroid cells culturedfrom a blood-type matched donor and expressing exogenous TP diluted in500 ml of saline solution via gravity drain over the course of 1 hr. Theprocedure is repeated monthly for 6 months. The patient's symptoms aremonitored over the duration of the treatment, including thymidine andthymine levels using HPLC.

The cultured erythroid cell population that over expresses thymidinephosphorylase is provided as a treatment for MNGIE.

A patient diagnosed as deficient for thymidine phosphorylase isintravenously administered single doses of 10̂9-10̂11 cells once a monthfor 6-12 months. During the course of treatment patients' thymidine andthymine levels are monitored with daily blood tests and relevant MNGIEsymptoms such as gastrointestinal behavior and cachexia are documented.

A population of 10̂11 erythroid cells expressing between 10K and 100Kcopies of thymidine phosphorylase per cell is stored in a transfusionbag with CPDA-1 and glycerol and stored at −80 C for up to 10 years.Upon treatment, the bag is thawed, centrifuged, and the cells removedand resuspended in saline for administration to a patient. Cells areintravenously administered with a 50 gauge needle at 5 ml/min at 37 C.

Example 82: Gaucher Disease

An in vitro assay is conducted to demonstrate the delivery ofβ-glucocerebrosidase (GC) to macrophages using GC-loaded, or expressing,erythroid cells. Successful delivery is indicative of potentialmechanistic action as a treatment for Gaucher's disease. Primarycultures of macrophages are prepared using a U937 cell line. Erythroidcells are loaded with GC and CFSE using transgene expression methods andstandard protocol for small molecule loading, washed with Alsever'ssolution and added to the macrophages on coverslips at a ratio of 10:1.Plates are centrifuged at 2600 g for 5 min and incubated at 37 C for 30min. Non-phagocytosed erythroid cells are lysed with hypotonic buffer.Macrophages are washed with PBS and stained with benzidine and Giemsa.The macrophages are then analyzed with FACS for internalized GC andCFSE, as well as accumulated ceramide levels using a diacylglycerol(DAG) kinase assay.

Erythroid cells from mice are washed with Alsever's solution and stainedwith PKH26 (Sigma Aldrich). Labeled, GC-loaded cells are injected intomice intraperitoneally. Four days after injection spleens are preparedfor microscopy and 12 micron sections are visualized using a fluorescentmicroscope. PKH26 is observed and quantified. In addition, GC-loadederythroid cells are administered and after 7 days circulating macrophagecell levels are quantified using FACS of respective CD antigens andcompared to levels in control mice. Ceramide levels in the macrophagepopulation are quantified using the DAG kinase assay.

A patient is diagnosed with Gaucher's disease according tocharacteristic symptoms such as; enlarged liver, anemia,thrombocytopenia, lung disease, arthritis, and genetic typing thatidentifies associated mutant genes. The patient is administered 10′11erythroid cells either loaded with or expressing GC. The cells arediluted in 500 ml of saline solution and are administered via gravitydrain over the course of 1 hr. The procedure is repeated monthly for 6months. The patient's symptoms are monitored over the duration of thetreatment, including macrophage counts, bleeding and thrombotic events,and macrophage ceramide levels.

Example 83: ALL-Asparaginase

Asparaginase activity is monitored in vitro using HPLC protocol todetect L-asparagine and aspartic acid levels. Approximately 10̂5erythroid cells expressing an exogenous, intracellular asparaginase,produced using the standard transfection protocol, are aliquoted into 1ml wells. 1 mM of asparagine is administered to each well and incubatedfor 1 hr. The cells are centrifuged and soluble protein is removed fromthe supernatant using cold methanol precipitation. The supernatantsamples are then run on an Agilent 1100 HPLC using a standard asparagineand aspartic acid curve to determine the relative amounts of amino acidand intracellular enzymatic activity compared to natural cells.

Asparaginase activity is monitored in vivo using HPLC protocol to detectasparagine and aspartic acid levels. An acute lymphocytic leukemia mousemodel created via insertion of a mutant NOTCH1 gene is treated withclodronate for 3 days prior to cell therapy administration (Haragushi,Mol. Cell Biol 2002). 100 ul of 10̂8 asparaginase expressing erythroidcells are administered via tail vein injection to the mouse model andblood samples are taken via a submandibular bleed at 10 min, 12 h, 24,h, 48 h, 72 h. The samples are analyzed using HPLC and asparagine andaspartic acid levels are tracked over time as well as with T cellproliferation behavior demonstrative of leukemia progression.

A patient diagnosed with ALL according to standard symptoms and somaticmutation analysis, including NOTCH1, RAS/PI3K/AKT deregulated signalingis intravenously administered 10̂11 erythroid cells cultured from ablood-type matched donor. The cells express exogenous, intracellularasparaginase diluted in 500 ml of saline solution and are administeredvia gravity drain over the course of 1 hr. The procedure is repeatedmonthly for 6 months. The patient's symptoms are monitored over theduration of the treatment, including asparagine and aspartic acid levelsusing HPLC. Proliferative leukemic cells are quantified usingimmunofluorescence of blood samples.

Example 84: Thrombotic Thrombocytopenic Purpura

An in vitro assay is conducted to demonstrate the activity of ADAMTS13expressed on a cultured erythroid cell's membrane. The ADAMTS13 assay isa FRET-inducible system that relies on a synthetic 73-amino-acidpeptide, FRETS-VWF73. Cleavage of this substrate between two modifiedresidues relieves the fluorescence quenching in the intact peptide.Incubation of FRETS-VWF73 with cultured erythroid cells, compared tonative erythrocytes, demonstrates quantitatively increased fluorescenceover time. Quantitative analysis is achieved within a 1-h period using a96-well format in commercial plate readers with common filters (Kokame,Br J Haematol, 2005).

The mechanistic ability of an ADAMTS13 expressing cultured erythroidcell is demonstrated using an NSG mouse model that is administeredclodronate for macrophage depletion. Recombinant, human Von Willebrandfactor (VWF) is injected at 10 mM via the tail vein. 10̂8 human erythroidcells expressing ADAMTS13 is subsequently injected and blood samples aretaken at 10 min, 1 hr, 4 hr, 8 hr, 24 hr. The serum is assayed for VWFcleavage using gel electrophoresis. Cleavage of the VWF by ADAMTS13takes place leading to a reduction in multimer sizes. This reduction isvisualized by agarose gel electrophoresis followed by Western blottingwith a peroxidase-conjugated anti-VWF antibody. The concentration ofADAMTS13 activity in the test sample is established by reference to aseries of diluted normal plasma samples.

A patient is diagnosed with thrombotic thrombocytopenia purpuraaccording to characteristic symptoms such as; thrombocytopenia,microangiopathic hemolytic anemia, neurologic symptoms, kidney failure,and genetic typing that identifies associated mutant genes. The patientis administered 10′11 erythroid cells expressing ADAMTS13 on thesurface. The cells are diluted in 500 ml of saline solution and areadministered via gravity drain over the course of 1 hr. The procedure isrepeated monthly for 6 months. The patient's symptoms are monitored overthe duration of the treatment, including VWF multimer levels, bleeding,and thrombotic events.

Example 85: Hemophilia B (FIX)

An in vitro assay is conducted to demonstrate the activity of Factor IX(FIX) expressed on a cultured erythroid cell's membrane. The Factor IXaassay protocol (activated Factor IX, BIOPHEN Factor IXa, Ref. A221812)is used to provide a quantitative chromogenic read out of a sample oferythroid cells. FIXa activity of the erythroid cells is compared tothat of both native erythrocytes and human plasma.

A mouse model of hemophilia B (Jackson Laboratories,B6.129P2-F9^(tm1Dws)/J) is immunosuppressed with cyclophosphamide andcleared of macrophages with clodronate. The mouse is then injected with10̂8 human erythroid cells expressing human FIX on the surface. The mouseis bled daily for 2 weeks via the tail vein and clotting time isrecorded. Results are compared to a negative control mouse model and apositive control model that receives a single dose of soluble FIX.

A patient is diagnosed with hemophilia B according to characteristicsymptoms such as; spontaneous bleeding, gastrointestinal tracthemorrhage, bruising, low circulating Factor IX levels, and genotypingthat confirms mutation in the X-linked gene. The patient is administered10′11 erythroid cells expressing FIX on the surface. The cells arediluted in 500 ml of saline solution and are administered via gravitydrain over the course of 1 hr. The procedure is repeated monthly for 6months. The patient's symptoms are monitored over the duration of thetreatment, including FIXa levels, spontaneous bleeding, and thromboticevents.

Example 85: Acute Lymphoblastic Leukemia

Erythroid cells expressing scFv against the leuekemic antigen, Wilms'tumor (WT1), are assayed in vitro for rosetting with primary sourced,WT1 positive, leukemia cells. Cells are cultivated at 3% hematocritusing McCoy's 5A medium enriched with 20% homologous serum, using themethod described by Russell et al. 2011 Blood 118(13):e74. The presenceof rosettes is detected and quantified using a novel Giemsa subvitalstaining methodology, modified from techniques applied in van Driessche,Leukemia 2005. The sampled culture suspension is stained with Giemsa(the final stain concentration is 5%) for 15 minutes. A small volume ofthis suspension (7.5 μl) is used to make a wet mount with 22×32 mm (0.17mm thickness) glass cover slip. The wet mount is examined immediatelywith light microscope under oil immersion magnification. The rosettingrate is determined by examining erythroid cells in McCoy's 5A mediumenriched with 20% homologous serum.

An NSG mouse model is treated with clodronate to eliminate itsmacrophage population. The mouse is injected with 10̂8 leukemic cellsthat are positive for WT1. A population of erythroid cells expressingmultiple copies of a scFv against WT1 on its surface is injected shortlythereafter and blood samples are taken at 10 min, 1 hr, 4 hr, 12 hr, 24hr, 48 hr time points. Samples are analyzed using FACS and erythroid-Bcell binding is quantified and compared to that of a positive controlerythrocyte infused mouse.

A patient diagnosed with acute lymphoblastic leukemia according tocharacteristic symptoms such as; fatigue, fever loss of weight, anemia,abnormal white blood cell count, and positive bone marrow biopsy. Thepatient is administered 10̂11 erythroid cells expressing anti-WT1 scFv onthe surface. The cells are diluted in 500 ml of saline solution and areadministered via gravity drain over the course of 1 hr. The procedure isrepeated monthly for 6 months. The patient's symptoms are monitored overthe duration of the treatment, including leukemic white blood cellcounts.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

Tables

TABLE 1 Erythroid Polypeptides and Non-Receiver Polypeptides ABO bloodgroups Stomatin Peters DAF Cromer Aquaporin 3 Tropomyosin RasmussenGerbich (GYPC) Aubergers Glucose transporter Reid CD47 Band 3 AdducinREIT Glycophorin A, B, C Basigin Rabphilin SARA Band 3 (AE3) C41 C1tetrahydrofolate Rhesus blood D group GYPB Ss synthase CD44 Vel groupAldolase C4A, C4B Chido, Rodgers C4 component of complement Cis AB Lanantigen Tropomodulin HLA Bg HLA class I Diego (Di) At antigen ArginaseRHAG Rh-associated Ammonium transport Colton antigen Jr antigen Creatinekinase glycoprotein Complement AnWj antigen B-Cam protein Colton (Co)Water Component 4 channel protein alpha(1,3) Sd antigen Rap1A ACHECartwright (Yt) fucosyltransferase Acetylcholinesterase CR1 BattyBennett-Goodspeed Glutathione transferase DAF Bilkes P antigen systemGlycophorin C Diego Wright (Wr) Rh blood group Aquaporin Duffy Box Xgantigen system Erythroblast associated membrane protein Hh/Bombayantigen Christiansen XK protein CD44 ii antigen alpha(1,2) Yt/Cartwrightantigen Synaptobrevin 2 fucosyltransferase system Indian blood group HJKCD58 Ribonuclease Kell HOFM Rh ABO glycosyl transferases Kidd JFV AnWjAdhesion CD59 receptor Lewis antigen JONEs Scianna CD44 Lutheran antigenJensen Radin MER2 MNS antigen system Katagiri Duodenal cytochrome DOKDombrock ADP- B ribosyltransferase Cost group Livesay DARC (Duffy)SEMA7A JMH Putative adhesion receptor Er group Milne CR1 Knops-McCoyUMOD Sda Tamm- Horsfall protein (uromodulin) Dematin Oldeide FP FamilyAnion exchanger channel protein (band 3, AE1) Indian (In) Annexin FamilyTweety Family CTL Family Kidd (Jk) Urea Bcl-2 Family UT Family DAACSFamily transporter FUT3 Lewis (Le) Bestrophin Family VIC Family DASSfamily Adenosine deaminase BNip3 Family AAAP Family DMT family OK OkaNeurothelin, CD20 Family transferrin receptor ENT Family putativeadhesion molecule LW Adhesion receptor CLIC Family c-KIT GPH Family FUT2Secretor (Se) Connexin Family Insulin receptors 1 & 2 GUP Family FUT1 Hhalpha CRAC-C Family Estrogen receptor LCT Family LU Lutheran (Lu) CtrFamily Dexamethasone MC family Adhesion receptor receptor P1Glycosyltransferase E-CIC Family JAK2 kinase MET Family XK Kx PutativeENaC Family ABC family MFS Family neurotransmitter transporter XG Xgformerly called GIC Family ArsAB family MOP Family PBDX MIC2 ICC FamilyF-ATPase Family MTC Family Hemoglobin Innexin Family IISP Family NCS2Family Ankyrin IRK-C Family MPT Family Nramp Family Spectrin LIC FamilyP-ATPase Family NSS Family KEL Kell (K, k, Kp, Js) MIP Family AE familyOAT Family Metalloproteinase Torkildsen MIT family APC Family OST FamilyRab 35 NSCC2 Family ArsB Family Oxa1 Family Ral A binding protein PCCFamily BASS Family PiT Family Zona pellucida binding Plamolipin FamilyCaCA Family PNaS Family protein Lyn B protein PLB Family CCC Family POTFamily KIaa1741 protein PLM Family CDF Family RFC Family DC38 PresenilinFamily CIC Family RND Family* Calciums transporting RIR-CaC Family CNTFamily SSS Family ATPase ACC Family TRIC Family CPA1 Family STRA6 FamilyAmt Family TRP-CC Family CPA2 Family SulP Family ZIP Family HCC FamilyNIPA Family N-MDE Family ATP-E Family LPI Family PPI Family Epo receptordsRNA-T Family MagT1 Family PPI2 Family MgtE Family

TABLE 2 Erythroid Cells Embryonic stem cells (ESC) Blastocytecolony-forming cells Cord blood stem cell (CD-SC) Burst-forming uniterythroid (BFU-E) CD34+ cells Megakaryocyte-erythroid progenitor (MEP)cell Hematopoietic stem cells (HSC) Erythroid forming colony unit(CFU-E) Spleen colony forming unit (CFU-S) Reticulocytes Common myeloidprogenitor (CMP) cells capable Erythrocytes of forming a granulocyte,erythrocyte, monocyte, or megakaryocyte (CFU-GEMM) Any cell of myeloidlineage Induced pluripotent stem cells (iPSC) ProerythroblastMesenchymal stem cell Polychromatophilic erythrocyte Polychromaticnormoblasts Normoblast Orthochromatic normoblasts

TABLE 3 Erythroid Promoters Promoter Gene beta globin promoter betaglobin 3′ beta-globin enhancer beta globin beta globin locus controlregion beta globin GATA-1 promoter GATA-1 GYPA promoter Glycophorin AHK1 promoter Hexokinase

TABLE 4 Sequences of Complement Receptor 14A. CR1 isoform S precursor, Homo sapiens NCBI Reference Sequence No. NP_000642.3   1 mgassprspe pvgppapglp fccggsllav vvllalpvaw gqcnapewlp farptnitde  61 fefpigtyln yecrpgysgr pfsiiclkns vwtgakdrcr rkscrnppdp vngmvhvikg 121 iqfgsqikys ctkgyrligs ssatciisgd tviwdnetpi cdripcglpp titngdfist 181 nrenfhygsv vtyrcnpgsg grkyfelyge psiyctsndd qvgiwsgpap qciipnkctp 241 pnvengilvs dnrslfslne vvefrcqpgf vmkgprrykc qalnkwepel pscsrvcqpp 301 pdvlhaertq rdkdnfspgq evfyscepgy dlrgaasmrc tpqgdwspaa ptcevkscdd 361 fmgqllngry lfpvnlqlga kvdfvcdegf qlkgssasyc vlagmeslwn ssvpvceqif 421 cpsppvipng rhtgkplevf pfgktvnytc dphpdrgtsf dligestirc tsdpqgngvw 481 sspaprcgil ghcqapdhfl faklktqtna sdfpigtslk yecrpeyygr pfsitcldnl 541 vwsspkdvck rkscktppdp vngmvhvitd iqvgsrinys cttghrligh ssaecilsgn 601 aahwstkppi cqripcglpp tiangdfist nrenfhygsv vtyrcnpgsg grkyfelyge 661 psiyctsndd qvgiwsgpap qciipnkctp pnvengilvs dnrslfslne vvefrcqpgf 721 vmkgprrykc qalnkwepel pscsrvcqpp pdvlhaertq rdkdnfspgq evfyscepgy 781 dlrgaasmrc tpqgdwspaa ptcevkscdd fmgqllngry lfpvnlqlga kvdfvcdegf 841 qlkgssasyc vlagmeslwn ssvpvceqif cpsppvipng rhtgkplevf pfgktvnytc 901 dphpdrgtsf dligestirc tsdpqgngvw sspaprcgil ghcqapdhfl faldktqtna 961 sdfpigtslk yecrpeyygr pfsitcldnl vwsspkdvck rkscktppdp vngmvhvitd1021 iqvgsrinys cttghrligh ssaecilsgn aahwstkppi cqripcglpp tiangdfist1081 nrenfhygsv vtyrcnpgsg grkyfelyge psiyctsndd qvgiwsgpap qciipnkctp1141 pnvengilvs dnrslfslne vvefrcqpgf vmkgprrykc qalnkwepel pscsrvcqpp1201 pdvlhaertq rdkdnfspgq evfyscepgy dlrgaasmrc tpqgdwspaa ptcevkscdd1261 fmgqllngry lfpvnlqlga kvdfvcdegf qlkgssasyc vlagmeslwn ssvpvceqif1321 cpsppvipng rhtgkplevf pfgkavnytc dphpdrgtsf dligestirc tsdpqgngvw1381 sspaprcgil ghcqapdhfl faklktqtna sdfpigtslk yecrpeyygr pfsitcldnl1441 vwsspkdvck rkscktppdp vngmvhvitd iqvgsrinys cttghrligh ssaecilsgn1501 tahwstkppi cqripcglpp tiangdfist nrenfhygsv vtyrcnlgsr grkyfelyge1561 psiyctsndd qvgiwsgpap qciipnkctp pnvengilvs dnrslfslne vvefrcqpgf1621 vmkgprrykc qalnkwepel pscsrvcqpp peilhgehtp shqdnfspgq evfyscepgy1681 dlrgaaslhc tpqgdwspea prcavkscdd flgqlphgry lfpinlqlga kvsfvcdegf1741 rikgssyshc vlygmrslwn nsvpvcehif cpnppailng rhtgtpsgdi pygkeisytc1801 dphpdrgmtf nligestirc tsdphgngvw sspaprcels vraghcktpe qfpfasptip1861 indfefpvgt slnyecrpgy fgkmfsiscl enlvwssved ncrrkscgpp pepfngmvhi1921 ntdtqfgstv nyscnegfrl igspsttclv sgnnytwdkk apiceiisce ppptisngdf1981 ysnnrtsfhn gtvvtyqcht gpdgeqlfel vgersiycts kddqvgvwss ppprcistnk2041 ctapevenai rvpgnrsfft lteiirfrcq pgfvmvgsht vqcqtngrwg pklphcsrvc2101 qpppeilhge htlshqdnfs pgqevfysce psydlrgaas lhctpqgdws peaprctvks2161 cddflgqlph grvllpinlq lgakvsfvcd egfrlkgrsa shcvlagmka lwnssvpvce2221 qifcpnppai lngrhtgtpf gdipygkeis yacdthpdrg mtfnligess irctsdpqgn2281 gvwsspaprc elsvpaacph ppkiqnghyi gghvslylpg mtisyicdpg yllvgkgfif2341 ctdqgiwsql dhyckevncs fplfmngisk elemkkvyhy gdyvtlkced gytlegspws2401 qcqaddrwdp plakctsrth dalivgtlsg tiffilliif lswiilkhrk gnnahenpke2461 vaihlhsqgg ssvhprtlqt neensrvlp (Seq. ID No. 1)4B. CR1 isoform F precursor, Homo sapiens NCBI Reference Sequence No. NP_000564.2   1 mgassprspe pvgppapglp fccggsllav vvllalpvaw gqcnapewlp farptnitde  61 fefpigtyln yecrpgysgr pfsiiclkns vwtgakdrcr rkscrnppdp vngmvhvikg 121 iqfgsqikys ctkgyrligs ssatciisgd tviwdnetpi cdripcglpp titngdfist 181 nrenfhygsv vtyrcnpgsg grkvfelvge psiyctsndd qvgiwsgpap qciipnkctp 241 pnvengilvs dnrslfslne vvefrcqpgf vmkgprrykc qalnkwepel pscsrvcqpp 301 pdvlhaertq rdkdnfspgq evfyscepgy dlrgaasmrc tpqgdwspaa ptcevkscdd 361 fmgqllngrv lfpvnlqlga kvdfvcdegf qlkgssasyc vlagmeslwn ssvpvceqif 421 cpsppvipng rhtgkplevf pfgktvnytc dphpdrgtsf dligestirc tsdpqgngvw 481 sspaprcgil ghcqapdhfl faklktqtna sdfpigtslk yecrpeyygr pfsitcldnl 541 vwsspkdvck rkscktppdp vngmvhvitd iqvgsrinys cttghrligh ssaecilsgn 601 aahwstkppi cqripcglpp tiangdfist nrenfhygsv vtyrcnpgsg grkvfelvge 661 psiyctsndd qvgiwsgpap qciipnkctp pnvengilvs dnrslfslne vvefrcqpgf 721 vmkgprrvkc qalnkwepel pscsrvcqpp pdvlhaertq rdkdnfspgq evfyscepgy 781 dlrgaasmrc tpqgdwspaa ptcevkscdd fmgqllngrv lfpvnlqlga kvdfvcdegf 841 qlkgssasyc vlagmeslwn ssvpvceqif cpsppvipng rhtgkplevf pfgkavnytc 901 dphpdrgtsf dligestirc tsdpqgngvw sspaprcgil ghcqapdhfl faldktqtna 961 sdfpigtslk yecrpeyygr pfsitcldnl vwsspkdvck rkscktppdp vngmvhvitd1021 iqvgsrinys cttghrligh ssaecilsgn tahwstkppi cqripcglpp tiangdfist1081 nrenfhygsv vtyrcnlgsr grkvfelvge psiyctsndd qvgiwsgpap qciipnkctp1141 pnvengilvs dnrslfslne vvefrcqpgf vmkgprrykc qalnkwepel pscsrvcqpp1201 peilhgehtp shqdnfspgq evfyscepgy dlrgaaslhc tpqgdwspea prcavkscdd1261 flgqlphgrv lfpinlqlga kvsfvcdegf rikgssvshc vlvgmrslwn nsvpvcehif1321 cpnppailng rhtgtpsgdi pygkeisytc dphpdrgmtf nligestirc tsdphgngvw1381 sspaprcels vraghcktpe qfpfasptip indfefpvgt slnyecrpgy fgkmfsiscl1441 enlvwssved ncrrkscgpp pepfngmvhi ntdtqfgstv nyscnegfrl igspsttclv1501 sgnnvtwdkk apiceiisce ppptisngdf ysnnrtsfhn gtvvtyqcht gpdgeqlfel1561 vgersiycts kddqvgvwss ppprcistnk ctapevenai rvpgnrsfft lteiirfrcq1621 pgfvmvgsht vqcqtngrwg pklphcsrvc qpppeilhge htlshqdnfs pgqevfysce1681 psvdlrgaas lhctpqgdws peaprctvks cddflgqlph grvllpinlq lgakvsfvcd1741 egfrlkgrsa shcvlagmka lwnssvpvce qifcpnppai lngrhtgtpf gdipygkeis1801 yacdthpdrg mtfnligess irctsdpqgn gvwsspaprc elsvpaacph ppkiqnghyi1861 gghvslylpg mtisyicdpg yllvgkgfif ctdqgiwsql dhyckevncs fplfmngisk1921 elemkkvyhy gdyvtlkced gytlegspws qcqaddrwdp plakctsrth dalivgtlsg1981 tiffilliif lswiilkhrk gnnahenpke vaihlhsqgg ssvhprtlqt neensrvlp(Seq. ID No. 2)4C. Predicted CR1 isoform X1, Homo sapiens, NCBI Reference Sequence No. XP_005273121.1   1 mclgrmgass prspepvgpp apglpfccgg sllavvvlla lpvawgqcna pewlpfarpt  61 nitdefefpi gtylnyecrp gysgrpfsii clknsvwtga kdrcrrkscr nppdpvngmv 121 hvikgiqfgs qikysctkgy rligsssatc iisgdtviwd netpicdrip cglpptitng 181 dfistnrenf hygsvvtyrc npgsggrkvf elvgepsiyc tsnddqvgiw sgpapqciip 241 nkctppnven gilysdnrsl fslnevvefr cqpgfvmkgp rrykcqalnk wepelpscsr 301 vcqpppdvlh aertqrdkdn fspgqevfys cepgydlrga asmrctpqgd wspaaptcev 361 kscddfmgql ingrylfpvn lqlgakvdfv cdegfqlkgs sasycvlagm eslwnssvpv 421 ceqifcpspp vipngrhtgk plevfpfgkt vnytcdphpd rgtsfdlige stirctsdpq 481 gngvwsspap rcgilghcqa pdhflfaklk tqtnasdfpi gtslkyecrp eyygrpfsit 541 cldnlvwssp kdvckrksck tppdpvngmv hvitdiqvgs rinyscttgh rlighssaec 601 ilsgnaahws tkppicqrip cglpptiang dfistnrenf hygsvvtyrc npgsggrkvf 661 elvgepsiyc tsnddqvgiw sgpapqciip nkctppnven gilysdnrsl fslnevvefr 721 cqpgfvmkgp rrvkcqalnk wepelpscsr vcqpppdvlh aertqrdkdn fspgqevfys 781 cepgydlrga asmrctpqgd wspaaptcev kscddfmgql ingrylfpvn lqlgakydfy 841 cdegfqlkgs sasycvlagm eslwnssvpv ceqifcpspp vipngrhtgk plevfpfgkt 901 vnytcdphpd rgtsfdlige stirctsdpq gngvwsspap rcgilghcqa pdhflfaklk 961 tqtnasdfpi gtslkyecrp eyygrpfsit cldnlvwssp kdyckrksck tppdpvngmv1021 hvitdiqvgs rinyscttgh rlighssaec ilsgnaahws tkppicqlcq pppdvlhaer1081 tqrdkdnfsp gqevfyscep gydlrgaasm rctpqgdwsp aaptcevksc ddfmgqllng1141 rylfpvnlql gakvdfvcde gfqlkgssas ycvlagmesl wnssvpvceq ifcpsppvip1201 ngrhtgkple vfpfgkavny tcdphpdrgt sfdligesti rctsdpqgng vwsspaprcg1261 ilghcqapdh flfaldktqt nasdfpigts lkyecrpeyy grpfsitcld nlvwsspkdv1321 ckrkscktpp dpvngmvhvi tdiqvgsrin yscttghrli ghssaecils gntahwstkp1381 picqripcgl pptiangdfi stnrenfhyg svvtyrcnlg srgrkvfelv gepsiyctsn1441 ddqvgiwsgp apqciipnkc tppnvengil vsdnrslfsl nevvefrcqp gfvmkgprrv1501 kcqalnkwep elpscsrvcq pppeilhgeh tpshqdnfsp gqevfyscep gydlrgaasl1561 hctpqgdwsp eaprcavksc ddflgqlphg rylfpinlql gakvsfvcde gfrlkgssvs1621 hcvlvgmrsl wnnsvpvceh ifcpnppail ngrhtgtpsg dipygkeisy tcdphpdrgm1681 tfnligesti rctsdphgng vwsspaprce lsvraghckt peqfpfaspt ipindfefpv1741 gtslnyecrp gyfgkmfsis clenlvwssv edncrrkscg pppepfngmv hintdtqfgs1801 tvnyscnegf rligspsttc lvsgnnvtwd kkapiceiis ceppptisng dfysnnrtsf1861 hngtvvtyqc htgpdgeqlf elvgersiyc tskddqvgvw ssppprcist nkctapeven1921 airvpgnrsf ftlteiirfr cqpgfvmvgs htvqcqtngr wgpklphcsr vcqpppeilh1981 gehtlshqdn fspgqevfys cepsydlrga aslhctpqgd wspeaprctv kscddflgql2041 phgrvllpin lqlgakvsfv cdegfrlkgr sashcvlagm kalwnssvpv ceqifcpnpp2101 ailngrhtgt pfgdipygke isyacdthpd rgmtfnlige ssirctsdpq gngvwsspap2161 rcelsvpaac phppkiqngh yigghvslyl pgmtisyicd pgyllvgkgf ifctdqgiws2221 qldhyckevn csfplfmngi skelemkkvy hygdyvtlkc edgytlegsp wsqcqaddrw2281 dpplakctsr thdalivgd sgtiffilli iflswiilkh rkgnnahenp kevaihlhsq2341 ggssvhprtl qtneensrvl p (Seq. ID No. 3)

TABLE 5 Targets General Classes of Targets Microbes Polypeptides DNAAmino Acids Fungi Toxins RNA Prions Bacteria Lipids Parasites CytokinesVirus Cells Cellular debris Complement-associated moleculesComplement-Related Targets Immune complexes C3dg C4a C6 Factor B C3dkC4b C7 Factor D C3e C2 C8 Properdin Bb C4bp C9 C3 membrane attackcomplex Mannose-Binding Lectin (MBL) C3a C1q MBL-Associated SerineProtease 1 (MASP1) C3b C1r MBL-Associated Serine Protease 2 (MASP2) iC3bC1s C5 C3c C4 C5a Infectious Disease-Related Targets LipopolysaccharidesCell invasion Intermedilysin Secreted effector protein protein sptP Zonaoccludens toxin Cholera enterotoxin Invasion protein SeeligeriolysinsipA Actin polymerization protein Cysteine protease Iota toxin Serineprotease RickA component Ia Actin polymerization protein CytolethalIvanolysin Shiga toxin RickA distending toxin Adenosine monophosphate-Cytolysin LepB Sphingomyelinase protein transferase vopS adenylatecyclase Cytotoxic Lethal factor Staphylokinase necrotizing factorAdenylate cyclase ExoY Cytotoxin Leukotoxin StreptokinaseADP-ribosyltransferase Dermonecrotic Listeriolysin Streptolysinenzymatic component toxin Aerolysin Deubiquitinase Microbial Streptopaincollagenase Alpha-toxin Diphtheria toxin Outer membrane Suilysin proteinIcsA autotransporter Alveolysin Enterohemolysin Panton-ValentineSuperantigen Leucocidin F Alveolysin Enterotoxin Perfringolysin T3SSsecreted effector EspF Anthrolysin O Epidermal cell Pertussis toxinTetanus toxin differentiation inhibitor Arp2/3 complex-activatingExoenzyme Phospholipase Tir protein rickA Binary ADP- ExotoxinPlasminogen TolC ribosyltransferase CDT toxin activator Botulinumneurotoxin G-nucleotide Pneumolysin Toxic shock syndrome exchange factortoxin C2 toxin, component II Guanine nucleotide Protective antigenZink-carboxypeptidase exchange factor sopE CagA Heat stable Proteinkinase Zink-carboxypeptidase enterotoxin Calmodulin-sensitiveIgA-specific serine Pyolysin Zn-dependent peptidase adenylate cyclaseendopeptidase autotransporter Cell cycle inhibiting factor Inositolphosphate RTX toxin phosphatase sopB Other Molecular Targets G-CSF IL3IL10 MIP1a GM-CSF IL4 IL12 MIP1b M-CSF IL5 IFNa TGFb IL1a IL6 IFNb TNFaIL1b IL7 IFNg TNFb IL2 IL8 Self-antibodies Non-self antibodies PRP PRPcPRPsc PRPres Lipid & Cell Targets Circulating tumor cells very lowdensity triglycerides Fatty acids lipid (VLDL) Metastases high densitychylomicrons Cholesterol lipoprotein Eukaryotic cells low densityapolipoproteins lipoprotein

TABLE 6 Diseases and Conditions Cancers Acute Colorectal cancerMacroglobulinemia, Pleuropulmonary lymphoblastic Waldenström Blastoma,leukaemia (ALL) Childhood Acute myeloid Craniopharyngioma, Male BreastCancer Pregnancy and leukaemia (AML) Childhood Breast CancerAdrenocortical Cutaneous T-Cell Lymphoma Malignant Fibrous PrimaryCentral Carcinoma Histiocytoma of Bone Nervous System and Osteosarcoma(CNS) Lymphoma AIDS-Related Ductal Carcinoma In Situ Melanoma ProstateCancer Kaposi Sarcoma (DCIS) AIDS-Related Embryonal Tumors, Merkel CellCarcinoma Rare cancers lymphoma Childhood Anal Cancer Endometrial CancerMesothelioma Rectal Cancer Appendix Cancer Ependymoma, ChildhoodMetastatic Squamous Renal cell Neck Cancer with carcinoma Occult PrimaryAstrocytomas, Epithelial cancer Midline Tract Renal Pelvis and ChildhoodCarcinoma Ureter, Transitional Involving NUT Gene Cell Cancer AtypicalEsophageal Cancer Molar pregnancy Retinoblastoma Teratoid/RhabdoidTumor, Childhood Basal Cell Esthesioneuroblastoma, Mouth andRhabdomyosarcoma Carcinoma Childhood oropharyngeal cancer Bile ductcancer Ewing sarcoma Multiple Endocrine Salivary Gland NeoplasiaSyndromes, Cancer Childhood Bladder cancer Extragonadal Germ CellMultiple Sarcoma Tumor Myeloma/Plasma Cell Neoplasm Bone cancerExtrahepatic Bile Duct Cancer Mycosis Fungoides Secondary cancers Bowelcancer Eye Cancer Myelodysplastic Sézary Syndrome Syndromes Brain StemGallbladder Cancer Myelodysplastic/ Skin Cancer Glioma,Myeloproliferative Childhood Neoplasms Brain tumours Gastric cancerMyeloproliferative Skin cancer (non Disorders, Chronic melanoma) Breastcancer Gastrointestinal Carcinoid Nasal Cavity and Small Cell Lung TumorParanasal Sinus Cancer Cancer Bronchial Tumors, Germ Cell TumorNasopharyngeal cancer Small Intestine Childhood Cancer BurkittGestational trophoblastic Neuroblastoma Soft Tissue Sarcoma Lymphomatumours (GTT) Cancer of Glioma Non-Hodgkin Squamous Cell unknown primaryLymphoma Carcinoma Cancer spread to Hairy cell leukaemia Non-Small CellLung Squamous Neck bone Cancer Cancer with Occult Primary, MetastaticCancer spread to Head and neck cancer Oesophageal cancer Stomach(Gastric) brain Cancer Cancer spread to Heart Cancer, Childhood OralCancer Stomach cancer liver Cancer spread to Hepatocellular (Liver)Cancer Oral Cavity Cancer T-Cell Lymphoma, lung Cutaneous - see MycosisFungoides and Sézary Syndrome Carcinoid Tumor Histiocytosis, LangerhansCell Oropharyngeal Cancer Testicular cancer Carcinoma of HodgkinLymphoma Osteosarcoma (Bone Throat Cancer Unknown Primary Cancer)Cardiac (Heart) Hypopharyngeal Cancer Osteosarcoma and Thymoma andTumors, Childhood Malignant Fibrous Thymic Carcinoma HistiocytomaCentral Nervous Intraocular Melanoma Ovarian Cancer Thyroid CancerSystem Atypical Teratoid/Rhabdoid Tumor, Childhood Central Nervous IsletCell Tumors, Pancreatic Pancreatic Cancer Transitional Cell SystemEmbryonal Neuroendocrine Tumors Cancer of the Renal Tumors, ChildhoodPelvis and Ureter Central Nervous Kidney cancer Pancreatic Unknownprimary System, Childhood Neuroendocrine Tumors cancer (Islet CellTumors) Cervical cancer Langerhans Cell Histiocytosis Papillomatosis,Ureter and Renal Childhood Pelvis, Transitional Cell Cancer Chordoma,Childhood Laryngeal Cancer Paraganglioma Urethral Cancer ChoriocarcinomaLeukemia Parathyroid Cancer Uterine Cancer, Endometrial Chronic Lip andOral Cavity Cancer Penile Cancer Uterine Sarcoma Lymphocytic Leukemia(CLL) Chronic myeloid Liver cancer Pharyngeal Cancer Vaginal cancerleukaemia (CML) Chronic Lobular Carcinoma In Situ PheochromocytomaVulvar Cancer Myeloproliferative (LCIS) Disorders Colon cancer LowMalignant Potential Pituitary Tumor Waldenström Tumor MacroglobulinemiaLymphoma Lung Cancer Plasma Cell Wilms Tumor Neoplasm/Multiple MyelomaComplement and Immune Complex-Related Diseases Age-relatedANCA-associated vasculitis Glomerulonephritis - MYH9-related macular(Includes Pauci-immune) sparse hair - disease degenerationtelangiectasis Atypical Anti-glomerular basement Goodpasture's sndromeNail-patella hemolytic uremic membrane disease syndrome syndrome(Goodpasture's) Autoimmune Arthus Reaction Granulomatosis withNail-patella-like hemolytic anemia polyangiitis (ANCA and renal diseaseWegeners) C1 inhibitor Asthma Guillain-Barre Nephritis deficiencysyndrome C1q deficiency Atypical hemolytic uremic Hemolytic angioedemaNon-amyloid syndrome (HAE) monoclonal immunoglobulin deposition diseaseC1r deficiency Autoimmune inner ear disease Henoch-SchonleinPauci-immune (AIED) Sensorineural hearing purpura glomerulonephritisloss C1s deficiency Autoimmune uveitis HIVICK Pediatric systemic lupuserythematosus C2 deficiency Autosomal dominant Hypersensitivty Piersonsyndrome intermediate Charcot-Marie- vasculitis Tooth disease type E C3deficiency Behçet disease Hypocomplementemic Polyarteritis urticarialvasculitis C4 deficiency Berger (IgA) Nephropathy Idiopathic membranouspolyarteritis nodosa glomerulonephritis C5 deficiency Buergers diseaseIdiopathic nephrotic Polymyalgia syndrome rheumatica C6 deficiencyCentral nervous system IgA nephropathy Polymyositis vasculitis (Berger'sdisease) C7 deficiency Choroiditis IgA nephropathy/vasculitisPolymyositis/ (Henoch-Schonlein dermatomyositis purpura) C8 deficiencyChronic demyelinating Immune Poststaphilococcal polyneuropathy (CIDP)thrombocytopenia glomerulonephritis C9 deficiency Churg-strauss syndromeImmunobullous diseases Poststeptococcal glomerulonephritis CD55deficiency Cogan's syndrome Immunotactoid or Primary fibrillarymembranoproliferative glomerulopathy glomerulonephritis CD59 deficiencyCollagen type III Infection-related Rapidly progressive glomerulopathyglomerulonephritis glomerulonephritis (Crescentic) Complement Congenitaland infantile Inflammatory Rapidly progressive Factor I nephroticsyndrome myopathies glomerulonephritis deficiency (RPGN) ComplementCongenital membranous Juvenile Rasmussen factor-H related nephropathydue to maternal dermatomyositis syndrome 1(CFHR1) anti-neutralendopeptidase deficiency alloimmunization ComplementCryoglobulinaemia/Cold Juvenile polymyositis Reactive arthritis factor-Hrelated agglutinin diease 3(CFHR3) deficiency CR3/CR4 Cryoglobulinemicvasculitis Kawasaki disease Relapsing defieciency polychondritis(leukocyte adhesion deficiency 1) Factor B Cutaneous vasculitisLipoprotein Renal amyloidosis deficiency glomerulopathy Factor DDemyelinating myopathies Lupus nephritis Reynolds syndrome deficiency(paraprotein associated) Factor H Denys-Drash syndrome Lupus nephropathyRheumatoid arthritis deficiency Factor I Dermatomyositis May Hegglinanomaly Sarcoidosis (Nesnier deficiency Boeck Schuamann Disease) Ficolin3 Dermatomyositis Membranoglomerular Schimke deficiency nephritisimmunoosseous dysplasia MASP2 Diabetic nephropathy MembranoproliferativeScleroderma deficiency glomerulonephritis MBL deficiency Drug-inducedimmune Membranoproliferative Sebastian syndrome complex vasculitisglomerulonephritis Type I (MPGN Type I) Non-alcoholic Eosinophilicgranulomatosis Membranoproliferative Secondary steatohepatitis withpolyangiitis (Churgg- glomerulonephritis Type amyloidosis Strauss) II(Dense Deposit Disease, MPGN Type II) Paroxysmal Epstein SyndromeMembranoproliferative Severe or recurring nocturnal glomerulonephritisType C diff colitis hemoglobinuria III (MPGN Type III) ProperdinEssential mixed Membranouse Sjogren's syndrome deficencycryoglobulinemia glomerulonephritis Action Familial Mediterranean feverMenieres disease Staphylococcal or myoclonus - renal streptococcalsepsis failure syndrome Acute respiratory Familial renal amyloidosisMicroscopic polyangiitis Stiff person disease syndrome syndrome(ARDS)/Severe acute respiratory syndrome (SARS) Acute serum Familialsteroid-resistant Minimal change disease Systemic lupus sicknessnephrotic syndrome with erythematosus sensorineural deafness Adult-onsetStill Farmer's lung Mixed connective tissue Systemic sclerosis diseasedisease Age-related Fechtner Syndrome Mostly large vessel Takayasuarteritis macular vasculitis degeneration AL amyloidosis Fibronectinglomerulopathy mostly mediu m vessel Toxic epidermal vasculitisnecrolysis (Stevens Johnson syndrome) Alport's Fibrosing alveolitisMostly small vessel Transplantation/reper- syndrome vsculitis fusion(solid organ) Alzheimer's Focal segmental glomerular Muckle-Wellssyndrome Vasculitis disease Amyloidosis (AL, Focal segmental Myastheniagravis Wegener's AA, MIDD, Other) glomerulosclerosis granulomatosisGiant cell arteritis Frasier syndrome Galloway-Mowat syndrome Type 1diabetes Myasthenia gravis Graves' disease Pernicious anemia Crohn'sdisease alopecia areata thrombocytopenic Primary biliary purpuracirrhosis Ulcerative colitis autoimmune hepatitis Guillain-BarrePsoriasis syndrome Inflammatory autoimmune deramtomyositis AutoimmuneRheumatoid arthritis bowel syndrome myocarditis Multiple sclerosisJuvenile idiopathic arthritis Autoimmune pemphigus Vitiligo EnzymeDeficiencies & Vascular Diseases 2,4-dienoyl-CoA Fabry disease (1:80,000to Isobutyryl-CoA Peripheral reductase 1:117,000) dehydrogenaseneuropathy deficiency 2-Methyl-3- Familial hypercholesterolemiaIsovaleric acidemia Peroxisomal hydroxy butyric (1:500) disorders(1:50,000; aciduria e.g., Zellweger syndrome, neonataladrenoleukodystrophy, Refsum's disease) 2-methylbutyryl- Familialmyocardial Lactase deficiency Phenylketonuria CoA dehydrogenaseinfarct/stroke (common) 3-hydroxy-3- Fatty acid oxidation disordersLesch-Nyhan syndrome Primary methylglutaryl (1:10,000) hyperoxaluria(HMG) aciduria 3-methylglutaconic Galactokinase deficiency Lipoproteinlipase Propionic acidemia aciduria deficiency (rare) 3-oxothiolaseGalactose epimerase long-chain 1-3- Recurrent emesis deficiencyhdroxyacyl-CoA (1:100,000) dehydrogenase 4-hydroxybutyric GalactosemiaLysinuric protein Short-chain acyl- aciduria intolerance (rare) CoAdehydrogenase 5,10- Galactosemia (1:40,000) Lysinuric proteinSucrase-isomaltase methylenetetrahydrofolate intolerance (rare)deficiency (rare) reductase deficiency (common) 5-OxoprolinuriaGaucher's disease Malonic acidemia Symptoms of (pyroglutamicpancreatitis aciduria) Abetalipoproteinemia Glutaric acidemia type IMaple syrup urine Transferase deficient (rare) disease galactosemia(Galactosemia type 1) Acute Glutaric acidemia Type II Medium chainacyl-CoA Trifunctional protein Intermittent dehydrogenase deficiencyPorphyria Alkaptonuria Glutathione Synthetase Medium/short chain L-Tyrosinemia type 1 Deficiency w/5-oxoprolinuria 3-hydroxy acyl-CoAdehydrogenase Argininemia Glutathione Synthetase Medum-chain ketoacyl-Tyrosinemia type 2 Deficiency w/o 5- coA thiolase oxoprolinuriaargininosuccinate Glycogenolysis disorders Metachromatic Tyrosinemiatype 3 aciduria (1:20,000) leukodystrophy (1:100,000) BenignGlycogenosis, type I Metachromatic Upward gaze hyperphenylalaninemia(1:70,000) leukodystrophy paralysis (1:100,000) beta ketothiolaseHemolytic anemia due to Methylmalonic acidemia Very long chaindeficiency adenylate kinase deficiency (Cbl C) acyl-CoA dehydrogenaseBiopterin cofactor Hemolytic anemia due to Methylmalonic acidemia WilsonDisease biosynthesis deficiency in Glucose 6 (Cbl D) defects phosphatedehydrogenase Biopterin cofactor Hemolytic anemia due to Methylmalonicacidemia Aicardi-Goutieres regeneration diphosphoglycerate mutase(vitamin b12 non- Syndrome (may be an defects deficiency responsive)allelic form of CLE) biotin- Hemolytic anemia due to Methylmalonicacidemia Cutaneous lupus unresponsive 3- erythrocyte adenosine w/0homocystinuria erythematosus methylcrotonyl- deaminase overproductionCoA carboxylase deficiency Carbamoyl Hemolytic anemia due toMethylmalonic aciduria Dermatitis phosphate glucophosphate isomerase andhomocystinuria herpetiformis synthetase deficiency Carnitine Hemolyticanemia due to Mitochondrial disorders hemophilia A acylcarnitineglutathione reductase (1:30,000) translocase deficiency CarnitineHemolytic anemia due to Mitochondrial disorders hemophilia Bpalmitoyltransferase glyceraldehyde-3-phosphate (1:30,000; e.g., Idehydrogenase deficiency cytochrome-c oxidase deficiency; MELASsyndrome; Pearson's syndrome [all rare]) Carnitine Hemolytic anemia dueto Mitochondrial disorders Idiopathic steroid palmitoyltransferasepyrimidine 5′ nucleotidase (1:30,000; e.g., Leigh sensitive nephrotic IIdeficiency disease, Kearns-Sayre syndrome (same as syndrome [rare])focal segmental glomerulaosclerosis) Carnitine uptake Hemolytic anemiadue to red Mitochondrial disorders Immune defect cell pyruvate kinasedeficiency (1:30,000; e.g., thrombocytopenic lipoamide purpuradehydrogenase deficiency [rare]) citrullinemia HHH syndrome (rare)Mitochondrial disorders Myasthenia gravis type I (1:30,000; e.g.,Pearson's syndrome [rare]) Citrullinemia homocysteinuria Multiplecarboxylase Oligoarticular type II (holocarboxylase juvenile arthritissynthetase) Congenital Homocystinuria (1:200,000) Multiple carboxylaseScleroderma disorders of deficiency (e.g., glycosylation (rare)holocarboxylase synthetase [rare]) and biotinidase deficiencies(1:60,000) D-2- hyperammonemia/ornithinemia/ Muscle Solar urticariahydroxyglutaricaciduria citrullinemia (ornithine cramps/spasticity(maybe protophyria transporter defect) erythema) D-2-Hyperlipoproteinemia, types I Myoadenylate Thrombotichydroxyglutaricaciduria and IV (rare) deaminase deficiencythrombocytopenic (rare) (1:100,000) purpura EnteropeptidaseHypermethioninemia due to Niemann-Pick disease, Tubulointerstitialdeficiency (rare) glycine N-methyltransferase type C (rare) nephritiswith deficiency Uveitis/ATIN Ethylmalonic Hypermethioninemia NonketoticVon willebrand encephalopathy encephalopathy due to hyperglycinemiadisease adenosine kinase deficiency Hyperprolinemia Infectious Diseases& agents Acinetobacter Dengue haemorrhagic fever Infection-inducedSepsis immune complex vasculitis Arcobacter butzleri Disseminatedinfection with Klebsiella Serratia infection - mycobacterium avium bloodinfection complex - blood infection Arcobacter cryaerophilus E. coliLeprosy/Hansen's Staphylococcus Aureus infection - blood diseaseinfection Arcobacter Enterobacter Malaria Stenotrophomonas infection -blood maltophilia - blood infection infection Bacteremia EnterococcusMeningococcus Streptococcal Group A invasive disease - blood infectionBacterial Glanders - blood infection Methicillin Resistant Streptococcusendocarditis Staphylococcus Aureus pneumoniae Campylobacter GonorrheaPseudomonas Streptococcus fetus infection - pyogenes blood infectionCampylobacter Hepatitis Rhodococcus equi - Trypanosomiasis jejuniinfection - blood infection blood infection Candida HumanImmunodeficiency Salmonella Yellow fever Virus Coagulase-negativeStaphylococcus

TABLE 7 Receivers General Classes of Receivers Ankyrin repeat proteinsFibronectins Lyases Antibodies Complement receptors GPI-linkedNanobodies polypeptides Aptamers Cyclic peptides HEAT repeat proteinsNucleic Acids ARM repeat proteins DARPins Hydrolases PolypeptidesCarbohydrates DNAses Kinases Single-chain variable fragments (scFv) Cellsurface receptors Enzymes Lipoproteins Tetratricopeptide repeat proteinsComplement-Related Receivers C1 inhibitor C4 binding protein CR3 FactorI C3 Beta chain CD59 CR4 Homologous restriction Receptor factor C3aR CR1Decay-accelerating Membrane cofactor factor (DAF) protein (MCP) C3eR CR2Factor H PRELP Enzymes triacylglycerol lipase bile-acid-CoA hydrolaseferuloyl esterase phosphatidate phosphatase (S)-methylmalonyl- bis(2-formyl-CoA hydrolase phosphatidylglycero- CoA hydrolaseethylhexyl)phthalate phosphatase esterase [acyl-carrier-protein]bisphosphoglycerate fructose- phosphatidylinositol phosphodiesterasephosphatase bisphosphatase deacylase [phosphorylase] Carboxylic-Esterfumarylacetoacetase phosphodiesterase I phosphatase Hydrolases1,4-lactonase carboxymethylenebutenolidase fusarinine-C phosphoglycerateornithinesterase phosphatase 11-cis-retinyl- cellulose-polysulfatasegalactolipase phosphoglycolate palmitate hydrolase phosphatase1-alkyl-2- cephalosporin-C gluconolactonase phosphoinositideacetylglycerophospho- deacetylase phospholipase C choline esterase2′-hydroxybiphenyl-2- cerebroside-sulfatase glucose-1- phospholipase A1sulfinate desulfinase phosphatase 2-pyrone-4,6- cetraxate benzylesteraseglucose-6- phospholipase A2 dicarboxylate phosphatase lactonase3′,5′-bisphosphate chlorogenate hydrolase glutathione phospholipase Cnucleotidase thiolesterase 3-hydroxyisobutyryl- chlorophyllaseglycerol-1- phospholipase D CoA hydrolase phosphatase 3′-nucleotidasecholinesterase glycerol-2- phosphonoacetaldehyde phosphatase hydrolase3-oxoadipate enol- choline-sulfatase glycerophosphocholinephosphonoacetate lactonase phosphodiesterase hydrolase 3-phytasecholoyl-CoA hydrolase Glycosidases, i.e. phosphonopyruvate enzymes thathydrolase hydrolyse O- and S- glycosyl compounds 4-hydroxybenzoyl-chondro-4-sulfatase glycosulfatase phosphoprotein CoA thioesterasephosphatase 4-methyloxaloacetate chondro-6-sulfatase GlycosylasesPhosphoric-diester esterase hydrolases 4-phytase citrate-lyasedeacetylase histidinol-phosphatase Phosphoric-monoester hydrolases4-pyridoxolactonase cocaine esterase hormone-sensitivePhosphoric-triester lipase hydrolases 5′-nucleotidase cutinaseHydrolysing N- phosphoserine glycosyl compounds phosphatase6-acetylglucose cyclamate Hydrolysing S- poly(3- deacetylasesulfohydrolase glycosyl compounds hydroxybutyrate) depolymerase 6-Cysteine endopeptidases hydroxyacylglutathione poly(3-phosphogluconolactonase hydrolase hydroxyoctanoate) depolymerasea-amino-acid esterase Cysteine-type hydroxybutyrate-polyneuridine-aldehyde carboxypeptidases dimer hydrolase esterasea-Amino-acyl-peptide D-arabinonolactonase hydroxymethylglutaryl-protein-glutamate hydrolases CoA hydrolase methylesteraseacetoacetyl-CoA deoxylimonate A-ring- iduronate-2-sulfatasequorum-quenching N- hydrolase lactonase acyl-homoserine lactonaseacetoxybutynylbithiophene dGTPase inositol-phosphate retinyl-palmitatedeacetylase phosphatase esterase acetylajmaline esterase dihydrocoumarinjuvenile-hormone Serine dehyrdatase or hydrolase esterase serinehydroxymethyl transferase acetylalkylglycerol Dipeptidases kynureninaseSerine endopeptidases acetylhydrolase acetylcholinesterase Dipeptidehydrolases L-arabinonolactonase serine- ethanolaminephosphatephosphodiesterase acetyl-CoA hydrolase Dipeptidyl-peptidaseslimonin-D-ring- Serine-type and tripeptidyl- lactonase carboxypeptidasespeptidases acetylesterase Diphosphoric-monoester lipoprotein lipaseS-formylglutathione hydrolases hydrolase acetylpyruvatedisulfoglucosamine-6- L-rhamnono-1,4- sialate O-acetylesterase hydrolasesulfatase lactonase acetylsalicylate dodecanoyl-[acyl- lysophospholipasesinapine esterase deacetylase carrier-protein] hydrolase acetylxylanesterase Endodeoxyribonucleases mannitol-1- Site specific producing 3′-phosphatase endodeoxyribonucleases: phosphomonoesters cleavage is notsequence specific acid phosphatase EndodeoxyribonucleasesMetallocarboxypeptidases Site-specific producing 5′-endodeoxyribonucleases phosphomonoesters that are specific for alteredbases. Acting on acid Endopeptidases of Metalloendopeptidases.Site-specific anhydrides to catalyse unknown catalyticendodeoxyribonucleases: transmembrane mechanism cleavage is sequencemovement of specific substances Acting on acid Endoribonucleasesmethylphosphothioglyc- sphingomyelin anhydrides to facilitate producing3′- erate phosphatase phosphodiesterase cellular and subcellularphosphomonoesters movement Acting on GTP to Endoribonucleasesmethylumbelliferyl- S-succinylglutathione facilitate cellular andproducing 5′- acetate deacetylase hydrolase subcellular movementphosphomonoesters Acting on phosphorus- Endoribonucleases thatmonoterpene e- steroid-lactonase nitrogen bonds are active with eitherlactone hydrolase ribo- or deoxyribonucleic acids and produce 3′-phosphomonoesters Acting on sulfur- Endoribonucleases that N- sterolesterase nitrogen bonds are active with either acetylgalactosamine-ribo- or 4-sulfatase deoxyribonucleic acids and produce 5′-phosphomonoesters actinomycin lactonase Enzymes acting on acid N-steryl-sulfatase anhydrides acetylgalactosamine- 6-sulfataseacylcarnitine hydrolase Enzymes Acting on N- succinyl-CoA carbon-carbonbonds acetylgalactosaminoglycan hydrolase deacetylase acyl-CoA hydrolaseEnzymes acting on N-acetylglucosamine- sucrose-phosphate carbon-nitrogenbonds, 6-sulfatase phosphatase other than peptide bonds acylglycerollipase Enzymes acting on N-sulfoglucosamine sugar-phosphatasecarbon-phosphorus sulfohydrolase bonds acyloxyacyl hydrolase Enzymesacting on oleoyl-[acyl-carrier- Sulfuric-ester carbon-sulfur bondsprotein] hydrolase hydrolases acylpyruvate hydrolase Enzymes Acting onOmega peptidases tannase ether bonds ADAMTS13 Enzymes acting onorsellinate-depside Thioester hydrolases halide bonds hydrolaseAdenosine deaminase Enzymes acting on oxaloacetase Thioether and peptidebonds trialkylsulfonium (peptidases) hydrolases adenylyl-[glutamate-Enzymes acting on palmitoyl[protein] Threonine ammonia ligase]phosphorus-nitrogen hydrolase endopeptidases hydrolase bondsADP-dependent Enzymes acting on palmitoyl-CoA thymidinemedium-chain-acyl- sulfur-nitrogen bonds hydrolase phosphorylase CoAhydrolase ADP-dependent short- Enzymes acting on pectinesterasetrehalose-phosphatase chain-acyl-CoA sulfur-sulfur bonds hydrolase ADP-Ether hydrolases. Peptidyl peptide triacetate-lactonase phosphoglyceratehydrolases phosphatase alkaline phosphatase ExodeoxyribonucleasesPeptidyl-amino-acid Triphosphoric- producing 5′- hydrolases monoesterhydrolases phosphomonoesters all-trans-retinyl- Exonucleases that arePeptidylamino-acid trithionate hydrolase palmitate hydrolase active witheither ribo- hydrolases or or deoxyribonucleic acylamino-acid acids andproduce 3′- hydrolases phosphomonoesters aminoacyl-tRNA Exonucleasesthat are Peptidyl-dipeptidases tropinesterase hydrolase active witheither ribo- or deoxyribonucleic acids and produce 5′- phosphomonoestersAminopeptidases Exoribonucleases phenylacetyl-CoA ubiquitinthiolesterase producing 3′- hydrolase phosphomonoesters arylesteraseExoribonucleases Phenylalanine UDP-sulfoquinovose producing 5′- ammonialyase synthase phosphomonoesters. arylsulfatase Factor IX Phenylalanineuricase hydroxylase Asparaginase Factor VIII pheophorbidaseuronolactonase Aspartic fatty-acyl-ethyl-ester phloretin hydrolasewax-ester hydrolase endopeptidases synthase b-diketone hydrolasephorbol-diester xylono-1,4-lactonase hydrolase

TABLE 8 Selected Diseases, Receivers and Targets Category DiseaseReceiver Target Amyloidoses AA Amyloidosis an an antibody-like binder toSerum amyloid A serum amyloid A protein or protein and amyloid serumamyloid P component placques Amyloidoses beta2 microglobulin an anantibody-like binder to Beta2 microglobulin or amyloidosis beta-2microglobulin or serum amyloid placques amyloid P component AmyloidosesLight chain amyloidosis an an antibody-like binder to Antibody lightchain or light chain, serum amyloid P amyloid placques component CellCancer an an antibody-like binder to a circulating tumor cell clearanceCD44 Cell Cancer an an antibody-like binder to a circulating tumor cellclearance EpCam Cell Cancer an an antibody-like binder to a circulatingtumor cell clearance Her2 Cell Cancer an an antibody-like binder to acirculating tumor cell clearance EGFR Cell Cancer (B cell) an anantibody-like binder to a cancerous B cell clearance CD20 Cell Cancer (Bcell) an an antibody-like binder to a cancerous B cell clearance CD19Clearance Antiphospholipid beta2-glycoprotein-1 pathogenic self- Absyndrome antibody against beta2- glycoprotein-1 Clearance Catastrophicbeta2-glycoprotein-1 pathogenic self- Ab antiphospholipid antibodyagainst beta2- syndrome glycoprotein-1 Clearance Cold agglutinin diseaseI/i antigen Pathogenic self- Ab antibody against I/i antigen ClearanceGoodpasture syndrome a3 NC1 domain of collagen pathogenic self- Ab (IV)antibody against a3 NC1 domain of Collagen (IV) Clearance ImmunePlatelet Glycoproteins (Ib-IX, pathogenic self- Ab thrombocytopeniaIIb-IIIa, IV, Ia-IIa) antibody against purpura platelet glycoproteinClearance Membranous Phospholipase A2 receptor pathogenic self- AbNephropathy antibody against phospholipase A2 receptor Clearance Warmantibody Glycophorin A, glycophorin B, pathogenic self- Ab hemolyticanemia and/or glycophorin C, Rh antibody against antigen glycophorinsand/or Rh antigen Complement Age-related macular a suitable complementactive complement degeneration regulatory protein Complement Atypicalhemolytic complement factor H, or a active complement uremic syndromesuitable complement regulatory protein Complement Autoimmune hemolytic asuitable complement active complement anemia regulatory moleculeComplement Complement Factor I Complement factor I, a suitable activecomplement deficiency complement regulatory protein ComplementNon-alcoholic a suitable complement active complement steatohepatitisregulatory molecule Complement Paroxysmal nocturnal a suitablecomplement active complement hemoglobinuria regulatory protein Enzyme3-methylcrotonyl-CoA 3-methylcrotonyl-CoA 3-hydroxyvalerylcarnitine,carboxylase deficiency carboxylase 3-methylcrotonylglycine (3-MCG) and3- hydroxyisovaleric acid (3-HIVA) Enzyme Acute IntermittentPorphobilinogen deaminase Porphobilinogen Porphyria Enzyme Acutelymphoblastic Asparaginase Asparagine leukemia Enzyme Acute lymphocyticAsparaginase Asparagine leukemia, acute myeloid leukemia Enzyme Acutemyeloblastic Asparaginase Asparagine leukemia Enzyme Adenine adenineInsoluble purine 2,8- phosphoribosyltransferasephosphoribosyltransferase dihydroxyadenine deficiency Enzyme Adenosinedeaminase Adenosine deaminase Adenosine deficiency EnzymeAfibrinogenomia FI enzyme replacement Enzyme Alcohol poisoning Alcoholdehydrogenase/oxidase Ethanol Enzyme Alexander's disease FVII enzymereplacement Enzyme Alkaptonuria homogentisate oxidase homogentisateEnzyme Argininemia Ammonia monooxygenase ammonia Enzymeargininosuccinate Ammonia monooxygenase ammonia aciduria Enzymecitrullinemia type I Ammonia monooxygenase ammonia Enzyme Citrullinemiatype II Ammonia monooxygenase ammonia Enzyme Complete LCATLecithin-cholesterol Cholesterol deficiency, Fish-eye acyltransferase(LCAT) disease, atherosclerosis, hypercholesterolemia Enzyme Cyanidepoisoning Thiosulfate-cyanide Cyanide sulfurtransferase Enzyme DiabetesHexokinase, glucokinase Glucose Enzyme Factor II Deficiency FII enzymereplacement Enzyme Familial hyperarginemia Arginase Arginine EnzymeFibrin Stabilizing factor FXIII enzyme replacement Def. Enzyme Glutaricacidemia type I lysine oxidase 3-hydroxyglutaric and glutaric acid(C5-DC), lysine Enzyme Gout Uricase Uric Acid Enzyme Gout -hyperuricemia Uricase Uric acid (Urate crystals) Enzyme Hageman Def.FXII enzyme replacement Enzyme Hemolytic anemia due to pyrimidine 5′nucleotidase pyrimidines pyrimidine 5′ nucleotidase deficiency EnzymeHemophilia A Factor VIII Thrombin (factor II a) or Factor X EnzymeHemophilia B Factor IX Factor XIa or Factor X Enzyme Hemophilia C FXIenzyme replacement Enzyme Hepatocellular Arginine deiminase Argininecarcinoma, melanoma Enzyme Homocystinuria Cystathionine B synthasehomocysteine Enzyme hyperammonemia/ Ammonia monooxygenase Ammoniaornithinemia/citrullinemia (ornithine transporter defect) EnzymeIsovaleric acidemia Leucine metabolizing enzyme leucine Enzyme Leadpoisoning d-aminolevulinate lead dehydrogenase Enzyme Lesch-Nyhansyndrome Uricase Uric acid Enzyme Maple syrup urine Leucine metabolizingenzyme Leucine disease Enzyme Methylmalonic acidemia methylmalonyl-CoAmutase methylmalonate (vitamin b12 non- responsive) Enzyme Mitochondrialthymidine phosphorylase thymidine neurogastrointestinalencephalomyopathy Enzyme Mitochondrial Thymidine phosphorylase Thymidineneurogastrointestinal encephalomyopathy (MNGIE) Enzyme Owren's diseaseFV enzyme replacement Enzyme p53-null solid tumor Serine dehyrdatase orserine serine hydroxymethyl transferase Enzyme Pancreatic Asparaginaseasparagine adenocarcinoma Enzyme Phenylketonuria Phenylalaninehydroxylase, Phenylalanine phenylalanine ammonia lyase Enzyme Primaryhyperoxaluria Oxalate oxidase Oxalate Enzyme Propionic acidemiaPropionate conversion enzyme? Proprionyl coA Enzyme Purine nucleosidePurine nucleoside Inosine, dGTP phosphorylase deficiency phosphorylaseEnzyme Stuart-Power Def. FX enzyme replacement Enzyme ThromboticADAMTS13 ultra-large von Thrombocytopenic willebrand factor Purpura(ULVWF) Enzyme Transferase deficient galactose dehydrogenaseGalactose-1-phosphate galactosemia (Galactosemia type 1) EnzymeTyrosinemia type 1 tyrosine phenol-lyase tyrosine Enzyme von Willebranddisease vWF enzyme replacement IC clearance IgA Nephropathy Complementreceptor 1 Immune complexes IC clearance Lupus nephritis Complementreceptor 1 immune complex IC clearance Systemic lupus Complementreceptor 1 immune complex erythematosus Infectious Anthrax (B.anthracis) an an antibody-like binder to B. anthracis infection B.anthracis surface protein Infectious C. botulinum infection an anantibody-like binder to C. botulinum C. botulinum surface proteinInfectious C. difficile infection an antibody-like binder to C.difficile C. difficile surface protein Infectious Candida infection anantibody-like binder to candida candida surface protein Infectious E.coli infection an antibody-like binder to E. coli E. coli surfaceprotein Infectious Ebola infection an antibody-like binder to EbolaEbola surface protein Infectious Hepatitis B (HBV) an antibody-likebinder to HBV HBV infection surface protein Infectious Hepatitis C (HCV)an antibody-like binder to HCV HCV infection surface protein InfectiousHuman an antibody-like binder to HIV HIV immunodeficiency virus envelopeproteins or CD4 or (HIV) infection CCR5 or Infectious M. tuberculosisinfection an antibody-like binder to M. tuberculosis M. tuberculosissurface protein Infectious Malaria (P. falciparum) an antibody-likebinder to P. falciparum infection P. falciparum surface protein LipidHepatic lipase Hepatic lipase (LIPC) Lipoprotein, deficiency,intermediate density hypercholesterolemia (IDL) LipidHyperalphalipoproteinemia 1 Cholesteryl ester transfer Lipoprotein, highprotein(CETP) density (HDL) Lipid hypercholesterolemia an antibody-likebinder to low- LDL density lipoprotein (LDL), LDL receptor Lipidhypercholesterolemia an antibody-like binder to high- HDL densitylipoprotein (HDL) or HDL receptor Lipid lipoprotein lipase lipoproteinlipase chilomicrons and very deficiency low density lipoproteins (VLDL)Lipid Lipoprotein lipase lipoprotein lipase (LPL) Lipoprotein, very lowdeficiency, disorders of density (VLDL) lipoprotein metabolism LysosomalAspartylglucosaminuria N-Aspartylglucosaminidase glycoproteins storage(208400) Lysosomal Cerebrotendinous Sterol 27-hydroxylase lipids,cholesterol, and storage xanthomatosis bile acid (cholestanol lipidosis;213700) Lysosomal Ceroid lipofuscinosis Palmitoyl-protein thioesterase-1lipopigments storage Adult form (CLN4, Kufs' disease; 204300) LysosomalCeroid lipofuscinosis Palmitoyl-protein thioesterase-1 lipopigmentsstorage Infantile form (CLN1, Santavuori-Haltia disease; 256730)Lysosomal Ceroid lipofuscinosis Lysosomal transmembrane lipopigmentsstorage Juvenile form (CLN3, CLN3 protein Batten disease, Vogt-Spielmeyer disease; 204200) Lysosomal Ceroid lipofuscinosis Lysosomalpepstatin-insensitive lipopigments storage Late infantile form peptidase(CLN2, Jansky- Bielschowsky disease; 204500) Lysosomal Ceroidlipofuscinosis Transmembrane CLN8 protein lipopigments storageProgressive epilepsy with intellectual disability (600143) LysosomalCeroid lipofuscinosis Transmembrane CLN6 protein lipopigments storageVariant late infantile form (CLN6; 601780) Lysosomal Ceroidlipofuscinosis Lysosomal transmembrane lipopigments storage Variant lateinfantile CLN5 protein form, Finnish type (CLN5; 256731) LysosomalCholesteryl ester storage lisosomal acid lipase lipids and cholesterolstorage disease (CESD) Lysosomal Congenital disorders ofPhosphomannomutase-2 N-glycosylated protein storage N-glycosylation CDGIa (solely neurologic and neurologic-multivisceral forms; 212065)Lysosomal Congenital disorders of Mannose (Man) phosphate (P)N-glycosylated protein storage N-glycosylation CDG Ib isomerase (602579)Lysosomal Congenital disorders of Dolicho-P-Glc: Man9GlcNAc2-N-glycosylated protein storage N-glycosylation CDG Ic PP-dolicholglucosyltransferase (603147) Lysosomal Congenital disorders ofDolicho-P-Man: N-glycosylated protein storage N-glycosylation CDG IdMan5GlcNAc2-PP- (601110) dolichol mannosyltransferase LysosomalCongenital disorders of Dolichol-P-mannose synthase N-glycosylatedprotein storage N-glycosylation CDG Ie (608799) Lysosomal Congenitaldisorders of Protein involved in mannose-P- N-glycosylated proteinstorage N-glycosylation CDG If dolichol utilization (609180) LysosomalCongenital disorders of Dolichyl-P-mannose: Man-7- N-glycosylatedprotein storage N-glycosylation CDG Ig GlcNAc-2-PP-dolichyl-α-6-(607143) mannosyltransferase Lysosomal Congenital disorders ofDolichyl-P-glucose: Glc-1-Man- N-glycosylated protein storageN-glycosylation CDG Ih 9-GlcNAc-2-PP-dolichyl-α-3- (608104)glucosyltransferase Lysosomal Congenital disorders ofα-1,3-Mannosyltransferase N-glycosylated protein storage N-glycosylationCDG Ii (607906) Lysosomal Congenital disorders ofMannosyl-α-1,6-glycoprotein- N-glycosylated protein storageN-glycosylation CDG IIa β-1,2-N- (212066) acetylglucosminyltransferaseLysosomal Congenital disorders of Glucosidase I N-glycosylated proteinstorage N-glycosylation CDG IIb (606056) Lysosomal Congenital disordersof GDP-fucose transporter-1 N-glycosylated protein storageN-glycosylation CDG IIc (Rambam-Hasharon syndrome; 266265 LysosomalCongenital disorders of β-1,4-Galactosyltransferase N-glycosylatedprotein storage N-glycosylation CDG IId (607091) Lysosomal Congenitaldisorders of Oligomeric Golgi complex-7 N-glycosylated protein storageN-glycosylation CDG IIe (608779) Lysosomal Congenital disorders ofUDP-GlcNAc: dolichyl-P N-glycosylated protein storage N-glycosylationCDG Ij NAcGlc phosphotransferase (608093) Lysosomal Congenital disordersof β-1,4-Mannosyltransferase N-glycosylated protein storageN-glycosylation CDG Ik (608540) Lysosomal Congenital disorders ofα-1,2-Mannosyltransferase N-glycosylated protein storage N-glycosylationCDG Il (608776) Lysosomal Congenital disorders ofα-1,2-Mannosyltransferase N-glycosylated protein storageN-glycosylation, type I (pre-Golgi glycosylation defects) LysosomalCystinosis Cystinosin (lysosomal cystine Cysteine storage transporter)Lysosomal Fabry's disease (301500) Trihexosylceramide α-globotriaosylceramide storage galactosidase Lysosomal Farber's diseaseCeramidase lipids storage (lipogranulomatosis; 228000) LysosomalFucosidosis (230000) α-L-Fucosidase fucose and complex storage sugarsLysosomal Galactosialidosis Protective protein/cathepsin A lysosomalcontent storage (Goldberg's syndrome, (PPCA) combined neuraminidase andβ-galactosidase deficiency; 256540) Lysosomal Gaucher's diseaseGlucosylceramide β- sphingolipids storage glucosidase Lysosomal Glutamylribose-5- ADP-ribose protein hydrolase glutamyl ribose 5- storagephosphate storage phosphate disease (305920) Lysosomal Glycogen storagedisease alpha glucosidase glycogen storage type 2 (Pompe's disease)Lysosomal GM1 gangliosidosis, Ganglioside β-galactosidase acidic lipidmaterial, storage generalized gangliosides Lysosomal GM2 activatorprotein GM2 activator protein gangliosides storage deficiency (Tay-Sachsdisease AB variant, GM2A; 272750) Lysosomal GM2 gangliosidosisGanglioside β-galactosidase gangliosides storage Lysosomal Infantilesialic acid Na phosphate cotransporter, sialic acid storage storagedisorder sialin (269920) Lysosomal Krabbe's disease Galactosylceramideβ- sphingolipids storage (245200) galactosidase Lysosomal Lysosomal acidlipase Lysosomal acid lipase cholesteryl storage deficiency (278000)esters and triglycerides Lysosomal Metachromatic Arylsulfatase Asulfatides storage leukodystrophy (250100) Lysosomal Mucolipidosis ML II(I- N-Acetylglucosaminyl-1- N-linked glycoproteins storage cell disease;252500) phosphotransfeerase catalytic subunit Lysosomal Mucolipidosis MLIII N-acetylglucosaminyl-1- N-linked glycoproteins storage(pseudo-Hurler's phosphotransfeerase polydystrophy) LysosomalMucolipidosis ML III Catalytic subunit N-linked glycoproteins storage(pseudo-Hurler's polydystrophy) Type III- A (252600) LysosomalMucolipidosis ML III Substrate-recognition subunit N-linkedglycoproteins storage (pseudo-Hurler's polydystrophy) Type III- C(252605) Lysosomal Mucopolysaccharidosis α-1-Iduronidaseglycosaminoglycans storage MPS I H/S (Hurler- Scheie syndrome; 607015)Lysosomal Mucopolysaccharidosis α-1-Iduronidase glycosaminoglycansstorage MPS I-H (Hurler's syndrome; 607014) LysosomalMucopolysaccharidosis Iduronate sulfate sulfatase glycosaminoglycansstorage MPS II (Hunter's syndrome; 309900) LysosomalMucopolysaccharidosis Heparan-S-sulfate sulfamidase glycosaminoglycansstorage MPS III (Sanfilippo's syndrome) Type III-A (252900) LysosomalMucopolysaccharidosis N-acetyl-D-glucosaminidase glycosaminoglycansstorage MPS III (Sanfilippo's syndrome) Type III-B (252920) LysosomalMucopolysaccharidosis Acetyl-CoA-glucosaminide N- glycosaminoglycansstorage MPS III (Sanfilippo's acetyltransferase syndrome) Type III-C(252930) Lysosomal Mucopolysaccharidosis N-acetyl-glucosaminine-6-glycosaminoglycans storage MPS III (Sanfilippo's sulfate sulfatasesyndrome) Type III-D (252940) Lysosomal Mucopolysaccharidosisα-1-Iduronidase glycosaminoglycans storage MPS I-S (Scheie's syndrome;607016) Lysosomal Mucopolysaccharidosis Galactosamine-6-sulfateglycosaminoglycans storage MPS IV (Morquio's sulfatase syndrome) TypeIV-A (253000) Lysosomal Mucopolysaccharidosis β-Galactosidaseglycosaminoglycans storage MPS IV (Morquio's syndrome) Type IV-B(253010) Lysosomal Mucopolysaccharidosis Hyaluronidase deficiencyglycosaminoglycans storage MPS IX (hyaluronidase deficiency; 601492)Lysosomal Mucopolysaccharidosis N-Acetyl galactosamine α-4-glycosaminoglycans storage MPS VI (Maroteaux- sulfate sulfatase(arylsulfatase Lamy syndrome; B) 253200) Lysosomal Mucopolysaccharidosisβ-Glucuronidase glycosaminoglycans storage MPS VII (Sly's syndrome;253220) Lysosomal Mucosulfatidosis Sulfatase-modifying factor-1sulfatides storage (multiple sulfatase deficiency; 272200) LysosomalNiemann-Pick disease Sphingomyelinase sphingomyelin storage type ALysosomal Niemann-Pick disease Sphingomyelinase sphingomyelin storagetype B Lysosomal Niemann-Pick disease NPC1 protein sphingomyelin storageType C1/Type D ((257220) Lysosomal Niemann-Pick disease Epididymalsecretory protein 1 sphingomyelin storage Type C2 (607625) (HE1; NPC2protein) Lysosomal Prosaposin deficiency Prosaposin sphingolipidsstorage (176801) Lysosomal Pycnodysostosis Cathepsin K kinins storage(265800) Lysosomal Sandhoff's disease; β-Hexosaminidase B gangliosidesstorage 268800 Lysosomal Saposin B deficiency Saposin B sphingolipidsstorage (sulfatide activator deficiency) Lysosomal Saposin C deficiencySaposin C sphingolipids storage (Gaucher's activator deficiency)Lysosomal Schindler's disease Type N-Acetyl-galactosaminidaseglycoproteins storage I (infantile severe form; 609241) LysosomalSchindler's disease Type N-Acetyl-galactosaminidase glycoproteinsstorage II (Kanzaki disease, adult-onset form; 609242) LysosomalSchindler's disease Type N-Acetyl-galactosaminidase glycoproteinsstorage III (intermediate form; 609241) Lysosomal Sialidosis (256550)Neuraminidase 1 (sialidase) mucopolysaccharides storage and mucolipidsLysosomal Sialuria Finnish type Na phosphate cotransporter, sialic acidstorage (Salla disease; 604369) sialin Lysosomal Sialuria French typeUDP-N-acetylglucosamine-2- sialic acid storage (269921) epimerase/N-acetylmannosamine kinase, sialin Lysosomal Sphingolipidosis Type IGanglioside β-galactosidase sphingolipids storage (230500) LysosomalSphingolipidosis Type II Ganglioside β-galactosidase sphingolipidsstorage (juvenile type; 230600) Lysosomal Sphingolipidosis TypeGanglioside β-galactosidase sphingolipids storage III (adult type;230650) Lysosomal Tay-Sachs disease; β-Hexosaminidase A gangliosidesstorage 272800 Lysosomal Winchester syndrome Metalloproteinase-2mucopolysaccharides storage (277950) Lysosomal Wolman's diseaselysosomal acid lipase lipids and cholesterol storage Lysosomalα-Mannosidosis α-D-Mannosidase carbohydrates and storage (248500), typeI (severe) glycoproteins or II (mild) Lysosomal β-Mannosidosisβ-D-Mannosidase carbohydrates and storage (248510) glycoproteins Toxicalpha hemolysin an antibody-like binder to alpha alpha hemolysinMolecule poisoning hemolysin Toxic antrax toxin poisoning anantibody-like binder to anthrax toxin Molecule anthrax toxin Toxicbacterial toxin-induced an antibody-like binder to bacterial toxinMolecule shock bacterial toxin Toxic botulinum toxin an antibody-likebinder to botulinum toxin Molecule poisoning botulinum toxin ToxicHemochromatosis (iron iron chelator molecular iron Molecule poisoning)Toxic Methanol poisoning Methanol dehdrogenase Methanol Molecule ToxicNerve gas poisoning Butyryl cholinesterase Sarin Molecule Toxic Priondisease caused by an antibody-like binder to prion Prion protein PRPMolecule PRP protein PRP Toxic Prion disease caused by an antibody-likebinder to prion Prion protein PRPc Molecule PRPc protein PRPc ToxicPrion disease caused by an antibody-like binder to prion Prion proteinPRPsc Molecule PRPsc protein PRPsc Toxic Prion disease cuased by anantibody-like binder to prion Prion protein PRPres Molecule PRPresprotein PRPres Toxic Sepsis or cytokine storm an antibody-like binder tocytokines Molecule cytokines or Duffy antigen receptor of chemokines(DARC) Toxic spider venom poisoning an antibody-like binder to spidervenom Molecule spider venom Toxic Wilson disease copper chelatormolecular copper Molecule

TABLE 9A Conjugation methods Zero-length x-linker Amine-sulfhydrylx-linker EDC SPDP, LC-SPDP, sulfo-LC- SPDP EDC plus sulfo NHS SMPT andsulfo-LC-SMPT CMC SMCC and sulfo-SMCC DCC MBS and sulfo-MBS DIC SIAB andsulfo-SIAB Woodward's reagent K SMPB and sulfo-SMPBN,N′-carbonyldiimidazole GMBS and sulfo-GMBS Schiff base + reductiveamination SIAX and SIAXX Homobifunctional NHS esters SIAC and SIACX DSPNPIA DTSSP Carbonyl-sulfydryl x-linker DSS MPBH BS{circumflex over ( )}3M2C2H DST PDPH Sulfo-DST amine-photoreactive x-linker BSOCOES NHS-ASA,Sulfo-NHS-ASA Sulfo-BSOCOES Sulfo-NHS-LC-ASA EGS SASD Sulfo-EGS HSAB andsulfo-HSAB DSG SANPAH and sulfo-SANPAH DSC ANB-NOS HomobifunctionalImidoesters SAND DMA SADP and sulfo-SADP DMP Sulfo-SAPB DMS SAED DTBPSulfo-SAMCA Sulfhydryl reactive x-linkers p-Nitrophenyl diazopyruvateDPDPB PNP-DTP BMH sulfhydryl-photoreactive x- linker Difluorobenzenederivatives ASIB DFDNB APDP DFDNPS Benzophenone-4-iodoacetamidePhotoreactive x-linker Benzophenone-4-maleimide BASEDCarbonyl-photoreactive x- linker Homobifunctional aldehydes ABHFormaldehyde Carboxylate-photoreactive x- linker Glutaraldehyde ASBAbis-epoxide arginine-photoreactive x-linker 1,4-butanediol diglycidylether APG Homobifunctional hydrazides Bioorthogonal reactions adipicacid dihydrazide Diels-alder reagent pairs carbohydrazideHydrazine-aldehyde reagent pairs Bis-diazonium derivative Boronic acidsalicylhydroxamate o-tolidine diazotized Click chemistry Bis-diazotizedbenzidine Staudinger ligation

TABLE 9B Enzymatic conjugation methods Sortase DD-transpeptidasePeptidyl transferase G-glutamyl transpeptidase D-glutamyl transpeptidaseFarnesyltransferase Prenyltranferase Dimethylallyltrans-transferaseGeranylgeranyl pyrophosphate synthase Dehydrodolichol diphosphatesynthase

TABLE 9C Chemistry of reactive groups Active hydrogen Amine reactionsThiol reactions Hydroxyl reactions reactions Isothyocyantes Haloacetyland alklhalide Epoxides and oxiranes Diazonium derivatives derivativesIsocyanates Maleimides Carbonyldiimidazole Mannich condensation Acylazides Aziridines N,N′0disuccinimidyl Iodination carbonate reactions NHSesters Acryloyl derivatives N-hydroxysuccinimidyl chloroformate Sulfonylchlorides Arylating agents Oxidation with periodate Aldehydes andThil-disulfide exchange Enzymatic oxidation Cycloaddition glyoxalsreagents reactions Epoxides and oxiranes Vinylsulfone derivatives Alkylhalogens Diels-Alder reaction Carbonates Metal-thiol dative bondsIsocyanates Complex formation with boronic acid derivatives

TABLE 10 Complement & Complement Regulatory Molecules Soluble moleculesAlternative Pathway Factor B Factor D Properdin C3 C3a C3b iC3b C3c C3dgC3dk C3e Bb Factor I Classical Pathway C1q C1r C1s C4 C4a C4b C2 C4bpLectin Pathway Mannose-Binding Lectin (MBL) MBL-Associated SerineProtease 1 (MASP1) MBL-Associated Serine Protease 2 (MASP2) LateComponents C5 C5a C6 C7 C8 C9 Receptors CR1 CR2 CR3 CR4 C3aR C3eRDecay-accelerating factor (DAF) Membrane cofactor protein (MCP) CD59 C3Beta chain Receptor Homologous restriction factor Control Proteins C1inhibitor C4 binding protein Factor I Factor H

1-148. (canceled)
 149. A method of making an erythroid cell comprising aphenylalanine ammonia lyase (PAL) polypeptide, comprising: i)introducing an exogenous nucleic acid encoding the PAL polypeptide intoan erythroid cell precursor; and ii) culturing the erythroid cellprecursor to provide an erythroid cell comprising the PAL polypeptide,wherein the erythroid cell comprising said PAL polypeptide is not ahypotonically loaded cells.
 150. The method of claim 149, wherein theerythroid cell comprises at least 1,000 copies of the PAL polypeptide.151. The method of claim 149, wherein the erythroid cell comprises atleast 10,000 copies of the PAL polypeptide.
 152. The method of claim149, wherein the erythroid cell comprises at least 25,000 copies of thePAL polypeptide.
 153. The method of claim 149, wherein the erythroidcell comprises at least 50,000 copies of the PAL polypeptide.
 154. Themethod of claim 149, wherein the erythroid cell further comprises asecond exogenous polypeptide which is surface-localized and comprises asubstrate transporter.
 155. The method of claim 149, wherein theerythroid cell is a reticulocyte.
 156. The method of claim 149, whereinthe erythroid cell is a mature erythrocyte.
 157. The method of claim149, wherein the erythroid cell lacks A and B antigens.
 158. The methodof claim 149, wherein the erythroid cell comprises fetal hemoglobin.159. The method of claim 149, wherein the erythroid cell exhibitssubstantially the same osmotic membrane fragility as an isolated,unmodified, cultured or uncultured erythroid cell.
 160. The method ofclaim 149, wherein the PAL polypeptide has a size from 500 amino acidsto 3000 amino acids.
 161. The method of claim 149, wherein the PALpolypeptide is glycosylated.
 162. The method of claim 149, wherein thePAL polypeptide does not substantially diffuse out of the erythroidcell.
 163. The method of claim 149, wherein the exogenous nucleic acidcomprises DNA or RNA.
 164. The method of claim 149, wherein theintroducing step comprises transduction, transfection, orelectroporation.
 165. The method of claim 164, wherein transfectioncomprises transfection with nanoparticles.
 166. The method of claim 149,wherein the culturing step comprises expanding the erythroid cellprecursor by at least 20,000-fold in culture.
 167. The method of claim149, which produces a population of erythroid cells wherein at least 70%of the cells are enucleated.
 168. The method of claim 149, whichproduces a population of erythroid cells wherein at least 70% of thecells are enucleated and comprise the PAL polypeptide.